CN117417452A - Monoclonal antibody of anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, kit containing same and detection method thereof - Google Patents
Monoclonal antibody of anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, kit containing same and detection method thereof Download PDFInfo
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- 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
- C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- 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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- 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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- 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
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Abstract
The application discloses a novel monoclonal antibody of an anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, an ELISA kit containing the antibody and a detection method thereof, and belongs to the technical field of biology. The antibodies described herein comprise an antibody heavy chain complementarity determining region and an antibody light chain complementarity determining region, the ELISA kit comprises an antigen and a pair of antibodies, wherein the antigen is human IFNAR1, the first antibody is QX006N, for binding to the coated antigen, and the second antibody is a monoclonal antibody of the anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody of the present application. The kit can be used for successfully completing the quantitative detection of the anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, has higher detection sensitivity and good detection specificity, and has good development and application prospects.
Description
Technical Field
The application belongs to the field of biotechnology, and in particular relates to a monoclonal antibody of an anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, an ELISA kit for quantitatively detecting the anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody by using the antibody, and a method for quantitatively detecting by using the kit.
Background
The I-type interferon (I-IFN) family plays an important role in both innate immunity and adaptive immunity, is mainly produced by plasmacytoid dendritic cells (pDC), and is combined with an I-IFN receptor (type I interferon-alpha/beta receptor, IFNAR) on the surface of cells, so that various genes are activated and expressed to further play various functions such as antiviral, antibacterial and immunoregulation. The I-IFN receptor consists of IFNAR1 and IFNAR2 chains. It has been reported that the expression of I-IFN regulatory genes in peripheral blood cells of patients with Systemic Lupus Erythematosus (SLE) is up-regulated, 60% -80% of SLE patients have high expression characteristics of I-IFN, and abnormally increased I-IFN promotes the occurrence and development of SLE by regulating the survival, activation and functions of cells related to the immune system, so blocking the I-IFN pathway may have therapeutic effects on SLE. The anti-human interferon alpha receptor 1 monoclonal antibody is an IgG4 kappa-type humanized monoclonal antibody which specifically binds to human interferon alpha receptor 1 (IFNAR 1); the parent antibody is obtained by screening B cells of New Zealand rabbits immunized by human IFNAR1, and the anti-human IFNAR1 monoclonal antibody is obtained after humanized modification of the parent rabbit antibody.
At present, the Aniffumab, which is a marketed co-target drug, is a fully humanized IgG1 kappa monoclonal antibody against human IFNAR1, which can bind to human IFNAR1 and block the signaling of all type I IFNs. Based on the main efficacy endpoint reached in all three clinical studies, anifloumab was shown to be useful in the treatment of SLE. The Jiangsu Xin biological medicine Co., ltd develops an anti-human IFNAR1 monoclonal antibody with the commodity code of QX006N as a drug, and the anti-human IFNAR1 monoclonal antibody is described in Chinese patent document CN 113278071B.
For drugs, pharmacokinetics is a method of applying the principle of dynamics and mathematical treatment, quantitatively describing the law of dynamic changes of drugs in vivo, and researching the processes of absorption, distribution, metabolism and excretion of drugs entering the human body in different ways. Thus, pharmacokinetic studies would be beneficial for the evaluation of drug safety and efficacy. Common analysis methods of biological samples include chromatography and immunoassay, the chromatography sensitivity is low, the test process is complicated, and the recovery rate is unstable; the immunological method is an analytical method based on specific antigen-antibody reaction, and is one of the most commonly used methods for detecting non-clinical and clinical biological samples at present. Because the anti-human IFNAR1 monoclonal antibody belongs to protein macromolecules, the method is suitable for measurement by using an enzyme-linked immunoassay method, and the rabbit anti-human IFNAR1 monoclonal antibody body is used as a second antibody to be combined with an enzyme-labeled detection antibody, the interference of irrelevant proteins in serum is reduced.
The indirect ELISA is to combine antigen onto ELISA plate, then add the antibody to be detected to combine with antigen, then add specific anti-antibody, finally add enzyme-labeled detection antibody and make use of substrate to generate color reaction. Thus, the specific combination of the antibody to be detected and the secondary antibody combines the combination of the enzyme-labeled detection antibody and the secondary antibody, not only plays a role in multistage amplification, but also provides greater flexibility, and fewer labeled antibodies are needed, so that the method is more economical. The method achieves the aim of detecting unknown antibody molecules and has the advantages of high sensitivity, high specificity, high stability and the like.
Disclosure of Invention
Based on the problems in the prior art, the application provides a monoclonal antibody of an anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, an improved indirect ELISA kit containing the antibody and developed based on an ELISA method, and the quantitative detection of the anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody is successfully completed by using the kit.
In particular, the present application relates to the following:
1. a monoclonal antibody directed against human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, wherein the monoclonal antibody comprises:
a) An antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein:
CDR-H1 has the amino acid sequence of the sequence SEQ ID NO 1 NSAMS or has at least 90% identity with SEQ ID NO 1,
CDR-H2 has the amino acid sequence of the sequence SEQ ID NO 2: IITDGAGTYYATWANG or has at least 90% identity with SEQ ID NO 2,
CDR-H3 has the amino acid sequence of SEQ ID NO 3: AAFGGSPLYLYDYGMDL or has at least 90% identity with SEQ ID NO 3;
b) An antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein:
CDR-L1 has the amino acid sequence of the sequence SEQ ID NO 4: QASQSIDSWLS or has at least 90% identity with SEQ ID NO 4,
CDR-L2 has the amino acid sequence of the sequence SEQ ID NO 5: ASTLTS or has at least 90% identity with SEQ ID NO 5,
CDR-L3 has the amino acid sequence of the sequence SEQ ID NO 6 QSNYYDPSNNYPNI or has at least 90% identity with SEQ ID NO 6.
2. The monoclonal antibody of item 1, wherein the monoclonal antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein:
HCVR has the sequence SEQ ID NO 7:
QSLEESGGRLVTPGTPLTLTCTVSGIDLSNSAMSWVRQAPGKGLEWLGIITDGAGTYYATWANGRFTISKTSSTVDLKITSPTTEDTATYFCASAAFGGSPLYLYDYGMDLWGPGTLVTVSS or at least 90% identical to SEQ ID NO 7;
LCVR has the amino acid sequence of SEQ ID NO 8: AEVVMTQTPASVEAAVGGTVTIKCQASQSIDSWLSWYQQKPGQPPVLLIYYASTLTSGVPSRFSGSGSGTEFTLTISDLECADAATYYCQSNYYDPSNNYPNIFGGGTEVVVK or has at least 90% identity to SEQ ID NO 8.
3. The monoclonal antibody according to item 1 or 2, wherein the monoclonal antibody comprises a heavy chain and a light chain, wherein:
the heavy chain amino acid sequence is the amino acid sequence of SEQ ID NO 9 QSLEESGGRLVTPGTPLTLTCTVSGIDLSNSAMSWVRQAPGKGLEWLGIITDGAGTYYATWANGRFTISKTSSTVDLKITSPTTEDTATYFCASAAFGGSPLYLYDYGMDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK or has at least 90% identity with SEQ ID NO 9;
the amino acid sequence of the light chain is the amino acid sequence of SEQ ID NO 10: AEVVMTQTPASVEAAVGGTVTIKCQASQSIDSWLSWYQQKPGQPPVLLIYYASTLTSGVPSRFSGSGSGTEFTLTISDLECADAATYYCQSNYYDPSNNYPNIFGGGTEVVVKGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC or has at least 90% identity with SEQ ID NO 10.
3-1. Use of the monoclonal antibody against human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody of item 1 in the preparation of a kit for detecting human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody.
The use according to item 3-1, wherein the kit comprises an antigen, a first antibody and a second antibody, the second antibody being a monoclonal antibody against the anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody according to item 1.
3-3 the use according to item 3-2, wherein the antigen is human IFNAR1 and the first antibody is QX006N.
The use according to item 3-2, wherein the antigen is immobilized to a solid support and the primary antibody binds to the antigen.
3-5 the use according to item 3-4, wherein the solid support is an ELISA plate.
The use according to item 3-2, wherein the kit further comprises: horseradish peroxidase-labeled goat anti-rabbit IgG Fc detection antibody, wash, substrate display solution, blocking solution/sample dilution, and stop solution.
The method according to any one of items 3-1 to 3-6, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.
3-8. the use according to any one of items 3-1 to 3-7, wherein the kit is a modified indirect enzyme-linked immunosorbent assay (ELISA) kit.
4. A kit for detecting an anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, characterized in that the kit comprises an antigen, a first antibody and a second antibody,
the antigen is human IFNAR1,
the first antibody is QX006N; comprising heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3, wherein:
the CDR-H1 of the first antibody QX006N has the amino acid sequence of SEQ ID NO 11:SYYMT, the CDR-H2 has the amino acid sequence of SEQ ID NO 12: VINVYGGTYYASWAKG, and the CDR-H3 has the amino acid sequence of SEQ ID NO 13: EDVAVYMAIDL; CDR-L1 has the amino acid sequence of SEQ ID NO 14: QASQSISNQLS, CDR-L2 has the amino acid sequence of SEQ ID NO 15:DASLAS, CDR-L3 has the amino acid sequence of SEQ ID NO 16: LGIYGDGADDGIA;
the second antibody is a monoclonal antibody against the anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody of any one of items 1-3.
5. The kit of item 4, wherein the antigen is immobilized to a solid support and the first antibody binds to the antigen.
6. The kit according to item 5, wherein the solid support is an ELISA plate.
7. The kit of any one of claims 4-6, wherein the kit further comprises: the goat anti-rabbit IgG Fc detection antibody marked by horseradish peroxidase, a washing liquid, a substrate display liquid, a dilution liquid/blocking liquid and a stopping liquid, wherein the washing liquid is preferably a mixed liquid of PBS solution and Tween20 (V: V=100:0.05), the substrate display liquid is TMB color development liquid, the stopping liquid is a mixed liquid of water and phosphoric acid (V: V=4:1), and the dilution liquid/blocking liquid is a mixed solution of PBS solution, tween20 and Proclin 300 (V: V: V=200:0.1:0.1).
8. The kit of any one of claims 4-7, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.
9. The kit according to any one of claims 4 to 8, wherein the kit is a modified indirect enzyme-linked immunosorbent assay (ELISA) kit.
10. A method for quantitatively detecting the content of anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibodies, wherein the antigen, primary antibody and secondary antibody of claim 4 are used for ELISA detection using a modified indirect enzyme-linked immunosorbent assay.
11. The method according to item 10, comprising the steps of:
fixing the antigen to a solid phase carrier, and coating and sealing;
adding a first antibody, incubating to enable the first antibody to react with the coated antigen, and then adding a second antibody for incubation;
adding a diluted goat anti-rabbit IgG Fc detection antibody marked by horseradish peroxidase, and incubating;
adding a substrate color development solution, adding a stop solution after light-shielding incubation, measuring an OD value, and preparing a standard curve;
substituting the OD value of the measured sample into an equation to calculate the content of the anti-human IFNAR1 monoclonal antibody in the measured sample.
In this application, the OD value is the OD value at 450 nm.
The application has the following beneficial effects:
firstly, taking a rabbit anti-human IFNAR1 monoclonal antibody as a second antibody, and establishing an ELISA method for quantitatively detecting the content of the anti-human IFNAR1 monoclonal antibody in serum; secondly, the effective quantitative range of the method established by the application is very wide, 30-1000 ng/ml, the lower limit of the quantitative of the anti-human IFNAR1 monoclonal antibody is 30ng/ml, and the sensitivity is high; thirdly, the method established by the application has good repeatability, and the variation coefficient of the repeated experiments among boards is 0.2-8.7% and less than 20%; fourth, the individual selectivity of the method established by the application is good, and 10/10 individuals meet the requirement that the accuracy and precision on the concentration level of the lower limit of quantification meet the acceptance range; fifth, the present application can be used to detect the content of anti-human IFNAR1 monoclonal antibodies in serum, and can be used to evaluate the pharmacokinetic profile of anti-human IFNAR1 monoclonal antibodies clinically, thereby evaluating their safety and effectiveness.
Drawings
FIG. 1A is a graph showing the results of binding of the monoclonal antibody prepared in example 1 to an anti-human IFNAR1 monoclonal antibody.
FIG. 1B is a graph showing the results of binding of the monoclonal antibody prepared in example 1 to human IgG.
FIG. 2 is a graph showing the results of the neutralization activity identification of the monoclonal antibody prepared in example 1.
FIG. 3 is a schematic representation of an ELISA method of the present application.
Fig. 4 is a standard curve four parameter fitting graph.
Detailed Description
The following examples are set forth in order to provide a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.
It should be noted that certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The specification and claims do not identify differences in terms of components, but rather differences in terms of the functionality of the components. As referred to throughout the specification and claims, the terms "include" or "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth the preferred embodiment for carrying out the present application, but is not intended to limit the scope of the present application in general, as the description proceeds. The scope of the present application is defined by the appended claims.
Technical and scientific terms used in the present specification have the same meaning as commonly understood by one of ordinary skill in the art, and if so conflict, the present specification will control.
Generally, terms used in the present specification have the following meanings.
In the present specification, "monoclonal antibody" means an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during production of monoclonal antibody preparations), which are typically present in minor amounts. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being derived from a substantially homogeneous population of antibodies, and is not to 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 are described herein, the monoclonal antibodies structurally comprising a heavy chain and a light chain.
In this specification, the CDR is an abbreviation for complement determine region (complementarity determining region), also called "hypervariable region". LCDR is an abbreviation for light chain hypervariable region and HCDR is an abbreviation for heavy chain hypervariable region. Typically, both the heavy and light chains of an antibody have 3 CDRs which together constitute the antigen-binding site of Ig. At this site, the antibody may be spatially complementary to the epitope. In the following, the use of the monoclonal antibodies claimed herein as "secondary antibodies" or "anti-antibodies" in analytical techniques specifically bind to the anti-human IFNAR1 monoclonal antibody, i.e. rely on the specific binding capacity of the CDR regions defined herein to specific epitopes on the human IFNAR1 monoclonal antibody.
In this specification, CVR is an acronym for "variable domain" and this definition is relative to "constant domain". The variable domains of both the heavy and light chains comprise three of the above "complementarity determining regions".
In the present specification, human IFNAR1 means a membrane protein derived from human. hIFNAR1 is a high affinity binding I-IFN transmembrane protein, can mediate I-IFN biological activity. The I-IFN family plays an important role in innate immunity and adaptive immunity, and is combined with an I-IFN receptor on the surface of a cell, so that various genes are activated and expressed, and then various functions such as antivirus, antibiosis, immunoregulation and the like are played. The monoclonal antibodies of the present application are neutralizing antibodies that specifically recognize human IFNAR1, which are capable of specifically binding to human IFNAR 1.
In the present specification, "identity" refers to the number of identical residues in two sequences when aligned for maximum correspondence in the context of amino acid sequences. There are many different algorithms known in the art that can be used to measure the percentage of amino acid identity (i.e., basic local alignment tool (Basic Local Alignment Tool) or BLAST). Default parameters for a particular program or algorithm are used unless otherwise indicated.
In the present specification, "anti-human IFNAR1 monoclonal antibody" means such a monoclonal antibody: which is capable of binding human IFNAR1 with sufficient affinity such that the monoclonal antibodies are useful for identifying/detecting human IFNAR1 and may also be used as antibody drugs, for example as drugs for the treatment of autoimmune diseases. Thus, the indirect 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 this specification, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein means that the binding pair (e.g., antibody and anti-antibodyOriginal) of the binding affinity of 1:1 interactions between members. The affinity of a molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K D ) And (3) representing. Affinity can be measured by common methods known in the art.
In the present specification, "enzyme-linked immunosorbent assay (EILSA)" refers to a detection method in which a free hetero protein is bound to a target protein bound to a solid carrier by utilizing the characteristic that an antibody molecule can specifically bind to an antigen molecule, and qualitative or quantitative analysis is performed using a specific label. The principle is as follows: the antigen or antibody can be physically adsorbed on the solid surface and maintain the immunological activity thereof; the antigen or antibody is capable of forming an enzyme conjugate with the enzyme via a covalent bond while maintaining the respective immunological or enzymatic activity; after binding the enzyme conjugate to the corresponding antigen or antibody, the occurrence of an immune response can be determined by a color reaction of the added substrate, the color reaction being in direct proportion to the amount of the corresponding antigen or antibody in the sample. According to the substance to be detected and the conditions of detection, various detection methods of different types can be designed, and the indirect method is the most common method for detecting antigens.
In this specification, a "standard curve" refers to a linear relationship between the content of a sample to be measured and a measurable (e.g., absorbance) value of an analysis method, to quantitatively analyze the sample to be measured by means of the same.
The present application relates to a monoclonal antibody against an anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, wherein the monoclonal antibody comprises: a) An antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein: CDR-H1 has the amino acid sequence of SEQ ID NO 1 NSAMS or has at least 90% identity with SEQ ID NO 1, CDR-H2 has the amino acid sequence of SEQ ID NO 2 IITDGAGTYYATWANG or has at least 90% identity with SEQ ID NO 2, CDR-H3 has the amino acid sequence of SEQ ID NO 3 AAFGGSPLYLYDYGMDL or has at least 90% identity with SEQ ID NO 3; b) An antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein: CDR-L1 has the amino acid sequence of SEQ ID NO 4: QASQSIDSWLS or at least 90% identity with SEQ ID NO 4, CDR-L2 has the amino acid sequence of SEQ ID NO 5:ASTLTS or at least 90% identity with SEQ ID NO 5, and CDR-L3 has the amino acid sequence of SEQ ID NO 6: QSNYYDPSNNYPNI or at least 90% identity with SEQ ID NO 1.
In a specific embodiment, the antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein: HCVR has the amino acid sequence of SEQ ID NO 7: QSLEESGGRLVTPGTPLTLTCTVSGIDLSNSAMSWVRQAPGKGLEWLGIITDGAGTYYATWANGRFTISKTSSTVDLKITSPTTEDTATYFCASAAFGGSPLYLYDYGMDLWGPGTLVTVSS or at least 90% identity to SEQ ID NO 7; LCVR has the amino acid sequence of SEQ ID NO 8: AEVVMTQTPASVEAAVGGTVTIKCQASQSIDSWLSWYQQKPGQPPVLLIYYASTLTSGVPSRFSGSGSGTEFTLTISDLECADAATYYCQSNYYDPSNNYPNIFGGGTEVVVK or has at least 90% identity to SEQ ID NO 8.
In a specific embodiment, the antibody comprises a heavy chain and a light chain, wherein:
the heavy chain amino acid sequence is the amino acid sequence of SEQ ID NO 9 QSLEESGGRLVTPGTPLTLTCTVSGIDLSNSAMSWVRQAPGKGLEWLGIITDGAGTYYATWANGRFTISKTSSTVDLKITSPTTEDTATYFCASAAFGGSPLYLYDYGMDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK or has at least 90% identity with SEQ ID NO 9; the amino acid sequence of the light chain is the amino acid sequence of SEQ ID NO 10: AEVVMTQTPASVEAAVGGTVTIKCQASQSIDSWLSWYQQKPGQPPVLLIYYASTLTSGVPSRFSGSGSGTEFTLTISDLECADAATYYCQSNYYDPSNNYPNIFGGGTEVVVKGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC or has at least 90% identity with SEQ ID NO 10.
In this specification, having at least 90% identity refers to having at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity.
The application relates to a kit for detecting an anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, wherein the kit comprises an antigen, a first antibody and a second antibody, wherein the antigen is human IFNAR1, and the first antibody is QX006N; comprising heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3, wherein: the CDR-H1 of the first antibody QX006N has the amino acid sequence of SEQ ID NO 11:SYYMT, the CDR-H2 has the amino acid sequence of SEQ ID NO 12: VINVYGGTYYASWAKG, and the CDR-H3 has the amino acid sequence of SEQ ID NO 13: EDVAVYMAIDL; CDR-L1 has the amino acid sequence of SEQ ID NO 14: QASQSISNQLS, CDR-L2 has the amino acid sequence of SEQ ID NO 15:DASLAS, CDR-L3 has the amino acid sequence of SEQ ID NO 16: LGIYGDGADDGIA; the second antibody is a monoclonal antibody of the anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody.
In a specific embodiment, the kit comprises an antigen that is human IFNAR1, a first antibody that is QX006N, and a second antibody; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO 17; and the amino acid sequence of the light chain variable region is shown as SEQ ID NO 18.
SEQ ID NO 17:
EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMTWVRQAPGKGLEWVSVINVYGGTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDVAVYMAIDLWGQGTLVTVSS
SEQ ID NO 18:
AIQMTQSPSSLSASVGDRVTITCQASQSISNQLSWYQQKPGKAPKLLIYDASSLASGVPSRFSGSRSGTKFTLTISSLQPEDFATYYCLGIYGDGADDGIAFGGGTKVEIK
The second antibody is a monoclonal antibody of the anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, and the amino acid sequence of the heavy chain variable region of the second antibody is shown as SEQ ID NO 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO 8.
SEQ ID NO 7:
QSLEESGGRLVTPGTPLTLTCTVSGIDLSNSAMSWVRQAPGKGLEWLGIITDGAGTYYATWANGRFTISKTSSTVDLKITSPTTEDTATYFCASAAFGGSPLYLYDYGMDLWGPGTLVTVSS
SEQ IN NO 8:
AEVVMTQTPASVEAAVGGTVTIKCQASQSIDSWLSWYQQKPGQPPVLLIYYASTLTSGVPSRFSGSGSGTEFTLTISDLECADAATYYCQSNYYDPSNNYPNIFGGGTEVVVK
In a specific embodiment, the kit comprises an antigen that is human IFNAR1, a first antibody that is QX006N, and a second antibody; the amino acid sequence of the heavy chain is shown as SEQ ID NO 19; the amino acid sequence of the light chain is shown as SEQ ID NO 20.
SEQ ID NO 19:
EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMTWVRQAPGKGLEWVSVINVYGGTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDVAVYMAIDLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO 20:
AIQMTQSPSSLSASVGDRVTITCQASQSISNQLSWYQQKPGKAPKLLIYDASSLASGVPSRFSGSRSGTKFTLTISSLQPEDFATYYCLGIYGDGADDGIAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
The second antibody is a monoclonal antibody of the anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, and the amino acid sequence of the second antibody containing a heavy chain is shown as SEQ ID NO 9; the amino acid sequence of the light chain is shown in SEQ ID NO 10.
SEQ ID NO 9:
QSLEESGGRLVTPGTPLTLTCTVSGIDLSNSAMSWVRQAPGKGLEWLGIITDGAGTYYATWANGRFTISKTSSTVDLKITSPTTEDTATYFCASAAFGGSPLYLYDYGMDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
SEQ ID NO 10:
AEVVMTQTPASVEAAVGGTVTIKCQASQSIDSWLSWYQQKPGQPPVLLIYYASTLTSGVPSRFSGSGSGTEFTLTISDLECADAATYYCQSNYYDPSNNYPNIFGGGTEVVVKGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
In a specific embodiment, the antigen is immobilized to a solid support and the first antibody binds to the antigen.
In a specific embodiment, the kit further comprises: horseradish peroxidase-labeled goat anti-rabbit IgG Fc detection antibody, wash, substrate display solution, blocking solution/sample dilution, and stop solution.
In a specific embodiment, the solid support is an elisa plate, preferably a 96-well elisa plate, specifically a transparent flat bottom non-sterile 96-well elisa plate from Thermo company. The substrate color development liquid is 3,3', 5' -tetramethyl benzidine (TMB) color development liquid; the wash solution was a mixture of PBS solution and tween20 (V: v=100:0.05); the diluent/blocking solution is a mixed solution of PBS solution, tween20 and Proclin 300 (V: V=200:0.1:0.1); the stop solution is a mixture of water and phosphoric acid (V: v=4:1).
In a specific embodiment, the ELISA kit comprises:
1) Solid phase carrier: the ELISA plate is 1 block;
2) Antigen: human IFNAR1;
3) Standard/quality control: a first antibody QX006N;
4) A second antibody: a monoclonal antibody directed against human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody;
5) Detection of antibodies: a goat anti-rabbit IgG Fc detection antibody labeled with horseradish peroxidase;
6) A substrate: 3,3', 5' -tetramethyl benzidine (TMB), 5 ml/tube, liquid A and liquid B have the same volume;
7) Coating antigen diluent: phosphate buffer (1 XPBS);
8) Washing liquid: a mixture of PBS solution and tween20 (V: v=100:0.05);
9) Dilution/blocking solution: a mixed solution of PBS solution, tween20 and Proclin 300 (V: v=200:0.1:0.1);
10 Termination liquid: water and phosphoric acid (V: v=4:1).
In a specific embodiment, the kit is an enzyme-linked immunosorbent assay (ELISA) kit. Preferably, the kit is a modified indirect enzyme-linked immunosorbent assay (ELISA) kit.
The present application also relates to a method for quantitatively detecting the content of an anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, wherein the method uses an antigen human IFNAR1, uses QX006N as a first antibody and binds to the antigen, uses a monoclonal antibody of the anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody as a second antibody, and uses a modified indirect enzyme-linked immunosorbent assay to perform ELISA detection, comprising the following steps: immobilizing an antigen on a solid phase carrier and coating and sealing; adding a first antibody for incubation; then adding a second antibody for incubation; adding a diluted goat anti-rabbit IgG Fc detection antibody marked by horseradish peroxidase, and incubating; adding a substrate color development solution, adding a stop solution after light-shielding incubation, measuring an OD value, and preparing a standard curve; substituting the OD value of the measured sample into an equation to calculate the content of the anti-human IFNAR1 monoclonal antibody in the measured sample.
In one embodiment, the ELISA kit described herein can be used to quantitatively detect the amount of anti-human IFNAR1 monoclonal antibody in a sample, comprising the steps of:
1) Coating: diluting human IFNAR1 to 1 μg/ml of coating working solution with 1 XPBS, adding into an ELISA plate (96-well Thermo ELISA plate) according to the dosage of 100 μl/well, and coating at 2-8deg.C overnight;
2) Closing: removing the coating working solution in the ELISA plate, washing the plate 3 times by using a washing solution (phosphate buffer solution (PBS) containing 0.05% Tween 20), adding a sealing solution according to the dosage of 200 mu l/hole, and sealing for about 2 hours at room temperature;
3) Sample adding: the standard product and the quality control product are uniformly diluted by 20 times by MRD with diluent; discarding the sealing liquid in the ELISA plate, washing the plate for 3 times by using washing liquid, adding the pretreated standard substance and quality control substance into the ELISA plate according to the dosage of 100 mu l/hole, and incubating for 2 hours at room temperature; wherein the standard substance and the quality control substance are QX006N;
4) Adding a secondary antibody: discarding standard substances and quality control substances in the ELISA plate, and washing the plate with washing liquid for 3 times; diluting the secondary antibody prepared in the example 1 into a secondary antibody working solution of 100ng/ml by using a diluent, adding the secondary antibody working solution into an ELISA plate according to the dosage of 100 mu l/hole, and incubating for about 1h at room temperature in a dark place;
5) Adding a detection antibody: discarding the secondary antibody working solution in the ELISA plate, washing the plate for 3 times by using a washing solution, diluting the goat anti-rabbit IgG antibody marked by horseradish peroxidase into a detection antibody working solution of 50ng/ml by using a diluting solution, adding the detection antibody working solution into the ELISA plate according to the dosage of 50 μl/hole, and incubating for about 1h at room temperature in a dark place;
6) Color development: mixing substrate solution A and solution B (3, 3', 5' -tetramethyl benzidine (TMB), 5 ml/tube, 1 tube each of solution A and solution B) completely; discarding the detection antibody working solution in the ELISA plate, washing the plate for 3 times by using a washing liquid, adding a substrate according to the dosage of 100 mu l/hole, and developing for 15-20min at room temperature in a dark place;
7) Termination and reading: and adding a termination solution into the ELISA plate according to the dosage of 100 mu l/hole to terminate the reaction, measuring the OD value of each hole in the ELISA plate at the wavelength of 450nm by using an ELISA, fitting a standard curve according to the OD value of a standard substance, substituting the OD value of a quality control sample into an equation, and calculating the concentration of the quality control sample.
Examples
EXAMPLE 1 preparation and purification of the second antibody
With anti-human IFNAR1 monoclonal antibody (F (ab) 2 ) Rabbit immunization is carried out as antigen, and non-medium antibody which can be combined with anti-human IFNAR1 monoclonal antibody, does not block the combination of the anti-human IFNAR1 monoclonal antibody and target human IFNAR1 thereof and does not generate cross reaction with other homotype human IgG is screened by B cell cloning technologyAnd a sex rabbit monoclonal antibody, the CDR sequences of which are SEQ ID NO 1-6, the variable region of the heavy chain is SEQ ID NO 7, the variable region of the light chain is SEQ ID NO 8, the heavy chain is SEQ ID NO 9, and the light chain is SEQ ID NO 10.
Example 2 characterization of the second antibody
1. Identification of binding Activity
The ELISA plate (purchased from Thermo) was coated with anti-human IFNAR1 monoclonal antibody and human IgG, respectively, disclosed in CN113278071B, and the monoclonal antibody prepared in example 1 was added after blocking and subjected to gradient dilution, and incubated at room temperature for 2 hours. After washing the plate, horseradish peroxidase-labeled goat anti-rabbit IgG antibody was added and incubated for 1 hour at room temperature. Finally, the plate is washed and developed, and OD is read 450 The absorbance is shown in FIG. 1A and FIG. 1B, wherein FIG. 1A is a graph showing the results of binding of the monoclonal antibody prepared in example 1 to the anti-human IFNAR1 monoclonal antibody, and FIG. 1B is a graph showing the results of binding of the monoclonal antibody prepared in example 1 to human IgG.
As can be seen from FIGS. 1A and 1B, the monoclonal antibody has better binding activity with the anti-human IFNAR1 monoclonal antibody, and does not cross react with homotype IgG.
2. Neutralization Activity assay
The ELISA plate was coated with human IFNAR1 as an antigen, and 100ng/ml of biotin-labeled CN113278071B was added thereto after blocking, and the mixture of the anti-human IFNAR1 monoclonal antibody disclosed in CN113278071B and the monoclonal antibody prepared in example 1 was subjected to gradient dilution, and incubated at room temperature for 2 hours. After washing the plate, horseradish peroxidase-labeled streptavidin antibody was added and incubated for 1 hour at room temperature. Finally, the plate is washed and developed, and OD is read 450 The absorbance at which the results are shown in FIG. 2, wherein 1 is the OD of the monoclonal antibody prepared in example 1 450 Absorbance at.
As can be seen from fig. 2, the monoclonal antibody does not block the binding of the anti-human IFNAR1 monoclonal antibody to its target human IFNAR1 and therefore does not possess neutralizing activity.
Example 3 construction of ELISA kit Using the "second antibody" prepared according to example 1
The antigen is human IFNAR1 and is immobilized on an elisa plate; the primary antibody can be combined with the coating antigen, and the preparation of the primary antibody and the performance measurement of the primary antibody are measured by the preparation method and the performance measurement method of CN 113278071B; the "secondary antibody" prepared in example 1 was used as the secondary antibody; the ELISA kit is constructed by adopting the method of figure 3 by taking goat anti-rabbit IgG antibody marked by horseradish peroxidase as a detection antibody.
The experimental process comprises the following steps:
1) Coating: diluting human IFNAR1 to 1 μg/ml of coating working solution with 1 XPBS, adding into an ELISA plate (96-well Thermo ELISA plate) according to the dosage of 100 μl/well, and coating at 2-8deg.C overnight;
2) Closing: removing the coating working solution in the ELISA plate, washing the plate 3 times by using a washing solution (phosphate buffer solution (PBS) containing 0.05% Tween 20), adding a sealing solution according to the dosage of 200 mu l/hole, and sealing for about 2 hours at room temperature;
3) Sample adding: diluting the standard and the quality control product by MRD (magnetic resonance imaging) for 20 times by using a diluent, discarding the sealing liquid in the ELISA plate, washing the plate for 3 times by using a washing liquid, adding the working liquid of the pretreated standard and quality control product into the ELISA plate according to the dosage of 100 mu l/hole, and incubating for 2 hours at room temperature; wherein the standard substance and the quality control substance are QX006N;
4) Adding a secondary antibody: discarding standard and quality control product working solution in the ELISA plate, washing the plate for 3 times by using washing liquid, diluting the secondary antibody prepared in the embodiment 1 to the secondary antibody working solution of 100ng/ml by using diluting liquid, adding the secondary antibody working solution into the ELISA plate according to the dosage of 100 mu l/hole, and incubating for about 1h at room temperature in a dark place;
5) Adding a detection antibody: discarding the secondary antibody working solution in the ELISA plate, washing the plate for 3 times by using a washing solution, diluting the goat anti-rabbit IgG antibody marked by horseradish peroxidase to a detection antibody working solution of 50ng/ml by using a diluting solution, adding the detection antibody working solution into the ELISA plate according to the dosage of 50 μl/hole, and incubating for about 1h at room temperature in a dark place;
6) Color development: mixing substrate solution A and solution B (3, 3', 5' -tetramethyl benzidine (TMB), 5 ml/tube, 1 tube each of solution A and solution B) completely; discarding the detection antibody working solution in the ELISA plate, washing the plate for 3 times by using a washing liquid, adding a substrate according to the dosage of 100 mu l/hole, and developing for 15-20min at room temperature in a dark place;
7) Termination and reading: and adding a termination solution into the ELISA plate according to the dosage of 100 mu l/hole to terminate the reaction, measuring the OD value of each hole in the ELISA plate at the wavelength of 450nm by using an ELISA, fitting a standard curve according to the OD value of a standard substance, substituting the OD value of a quality control sample into an equation, and calculating the concentration of the quality control sample.
Wherein, the reagent information is:
1 XPBS: 23.48g of PBS powder was dissolved in 2000ml of ultra pure water;
washing liquid: 500 μl Tween20 was added per 1000ml of 1 XPBS;
dilution/blocking solution: 2.5g BSA was weighed, dissolved in 500ml 1 XPBS, added with 250. Mu.l Tween20 and 250. Mu.l Proclin 300, and thoroughly mixed;
preparing TMB color development liquid: taking equal volumes of the solution A and the solution B, fully and uniformly mixing the solution A and the solution B after the solution A and the solution B reach room temperature, and preparing the solution from light to the spot;
stop solution: 100ml H 3 PO 4 Adding to 400ml of ultrapure water;
example 4 establishment of ELISA method for quantitative determination of anti-human IFNAR1 monoclonal antibody content in serum
1. Establishment of standard curve of ELISA method for quantitatively detecting content of anti-human IFNAR1 monoclonal antibody in serum
Anti-human IFNAR1 monoclonal antibodies were diluted to 15.625, 31.25, 62.5, 125, 250, 500, 1000ng/ml with human pooled serum (equal volumes of 10 individual blank serum) and subjected to an enzyme linked immunoassay according to example 3 under defined optimal experimental conditions. Four parameter curves were plotted with the concentrations of the detected anti-human IFNAR1 monoclonal antibodies on the abscissa and the OD450 values on the ordinate, as shown in fig. 4, and recovery rates were calculated, and the results thereof are shown in table 1.
Table 1: method standard curve establishment of recovery rate statistics of each concentration point
。
Curve equation: fit: y= (0.051-3.752)/(1+ (x/560.7)/(1.534) + 3.752)
Note that: * Marked as anchor point, recovery rate is not required
As can be seen from FIG. 4, the concentration of the anti-human IFNAR1 monoclonal antibody is within the range of 30-1000 ng/ml, and the curve has better recovery rate.
As can be seen from Table 1, the standard concentration of the ELISA method established in the present application is 15.625 (lower anchor point), 31.25, 62.5, 125, 250, 500, 1000ng/ml. The quantitative range of the method is 30-1000 ng/ml, and 15.625ng/ml is used as an anchor point for improving the fitting of standard curves (the recovery rate is not required).
2. Methodological research of enzyme-linked immunoassay method for quantitatively detecting content of anti-human IFNAR1 monoclonal antibody in serum
2.1 quantitative Range
The standard curves of 6 independent batches were evaluated by different laboratory personnel on different days using established ELISA methods for quantitative detection of anti-human IFNAR1 monoclonal antibody content in serum, and the results are shown in Table 2. It can be seen from Table 2 that, except for the anchor points, the concentration levels of LLOQ and ULOQ: the accuracy RE% between batches is within the range of-0.5% -3.3%, and the precision CV% is within the range of 0.2% -8.7%. 1000ng/mL and 30ng/mL can be used as the upper and lower quantitative limits of the method.
Table 2: quantitative scope of method study
。
2.2 accuracy and precision
The 6 independent batches of quality control samples (including LLOQ, LQC, MQC, HQC, ULOQ) were evaluated by an established ELISA method for quantitatively detecting the anti-human IFNAR1 monoclonal antibody content in serum, and the results are shown in Table 3.
As can be seen from table 3, at LLOQ and ULOQ concentration levels: the accuracy RE% between batches is within the range of-7.3% to-4.2%, and the precision CV% is within the range of 6.0% to 10.5%. At QC concentration level: the accuracy RE% between batches is within the range of-5.6% -1.0%, and the precision CV% is within the range of 4.4% -7.8%.
Table 3: method accuracy and precision study
。
2.3 Selectivity
The results of the established ELISA assay for quantitative detection of anti-human IFNAR1 monoclonal antibody content in serum were examined by adding LLOQ concentration levels of anti-human IFNAR1 monoclonal antibody to at least 10 individual sources of blank matrix, as shown in Table 4.
As can be seen from Table 4, the response values of the blank samples of 10 individual sources are lower than LLOQ, and the accuracy RE% in the batch of the quality control samples of the LLOQ level of 10 individual sources is in the range of-5.2% -7.4%.
Table 4: method selectivity study
。
EXAMPLE 5 content determination of anti-human IFNAR1 monoclonal antibody in serum Using ELISA kit constructed in example 3 and method constructed in example 4
Diluting an anti-human IFNAR1 monoclonal antibody sample to be detected by MRD 20 times by using a diluent, and diluting by a proper multiple by using 5% serum diluent to ensure that the diluent falls into a standard curve quantitative range; the content of the anti-human IFNAR1 monoclonal antibody in serum was determined using the ELISA kit constructed in example 3 and the method constructed in example 4, which can accurately measure the anti-human IFNAR1 monoclonal antibody.
In summary, the enzyme-linked immunoassay method for quantitatively detecting the content of the anti-human IFNAR1 monoclonal antibody in serum provided by the application is that a solid-phase carrier is formed by coating an ELISA plate with the human IFNAR1, the solid-phase carrier is combined with the anti-human IFNAR1 monoclonal antibody in a biological sample to be detected, and then a solid-phase antigen-antibody-ELISA antibody compound is formed by a specific secondary antibody and a detection antibody. The second antibody (secondary antibody) obtained by screening has strong specificity, so the method established in the application has strong specificity, high sensitivity and good precision and accuracy.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application in any way. Any person skilled in the art may make variations or modifications to the equivalent embodiments using the teachings disclosed above. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present application still fall within the protection scope of the technical solution of the present application.
Claims (11)
1. A monoclonal antibody directed against an anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, wherein the monoclonal antibody comprises:
a) An antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein:
CDR-H1 has the amino acid sequence of the sequence SEQ ID NO 1 NSAMS or has at least 90% identity with SEQ ID NO 1,
CDR-H2 has the amino acid sequence of the sequence SEQ ID NO 2: IITDGAGTYYATWANG or has at least 90% identity with SEQ ID NO 2,
CDR-H3 has the amino acid sequence of SEQ ID NO 3: AAFGGSPLYLYDYGMDL or has at least 90% identity with SEQ ID NO 3;
b) An antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein:
CDR-L1 has the amino acid sequence of the sequence SEQ ID NO 4: QASQSIDSWLS or has at least 90% identity with SEQ ID NO 4,
CDR-L2 has the amino acid sequence of the sequence SEQ ID NO 5: ASTLTS or has at least 90% identity with SEQ ID NO 5,
CDR-L3 has the amino acid sequence of the sequence SEQ ID NO 6 QSNYYDPSNNYPNI or has at least 90% identity with SEQ ID NO 6.
2. The monoclonal antibody of claim 1, wherein the monoclonal antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein:
HCVR has the sequence SEQ ID NO 7:
QSLEESGGRLVTPGTPLTLTCTVSGIDLSNSAMSWVRQAPGKGLEWLGIITDGAGTYYATWANGRFTISKTSSTVDLKITSPTTEDTATYFCASAAFGGSPLYLYDYGMDLWGPGTLVTVSS or at least 90% identical to SEQ ID NO 7;
LCVR has the sequence SEQ ID NO 8:
AEVVMTQTPASVEAAVGGTVTIKCQASQSIDSWLSWYQQKPGQPPVLLIYYASTLTSGVPSRFSGSGSGTEFTLTISDLECADAATYYCQSNYYDPSNNYPNIFGGGTEVVVK or at least 90% identical to SEQ ID NO 7.
3. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody comprises a heavy chain and a light chain, wherein:
the heavy chain amino acid sequence is the amino acid sequence of SEQ ID NO 9 QSLEESGGRLVTPGTPLTLTCTVSGIDLSNSAMSWVRQAPGKGLEWLGIITDGAGTYYATWANGRFTISKTSSTVDLKITSPTTEDTATYFCASAAFGGSPLYLYDYGMDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK or has at least 90% identity with SEQ ID NO 9;
the amino acid sequence of the light chain is the amino acid sequence of SEQ ID NO 10: AEVVMTQTPASVEAAVGGTVTIKCQASQSIDSWLSWYQQKPGQPPVLLIYYASTLTSGVPSRFSGSGSGTEFTLTISDLECADAATYYCQSNYYDPSNNYPNIFGGGTEVVVKGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC or has at least 90% identity with SEQ ID NO 10.
4. A kit for detecting an anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody, wherein the kit comprises an antigen, a first antibody and a second antibody,
the antigen is human IFNAR1;
the first antibody is QX006N; comprising heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3, wherein:
the CDR-H1 of the first antibody QX006N has the amino acid sequence of SEQ ID NO 11:SYYMT, the CDR-H2 has the amino acid sequence of SEQ ID NO 12: VINVYGGTYYASWAKG, and the CDR-H3 has the amino acid sequence of SEQ ID NO 13: EDVAVYMAIDL; CDR-L1 has the amino acid sequence of SEQ ID NO 14: QASQSISNQLS, CDR-L2 has the amino acid sequence of SEQ ID NO 15:DASLAS, CDR-L3 has the amino acid sequence of SEQ ID NO 16: LGIYGDGADDGIA;
the second antibody is a monoclonal antibody against an anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibody according to any one of claims 1 to 3.
5. The kit of claim 4, wherein the antigen is immobilized to a solid support and the first antibody binds to the antigen.
6. The kit of claim 5, wherein the solid support is an elisa plate.
7. The kit of any one of claims 4-6, wherein the kit further comprises: horseradish peroxidase-labeled goat anti-rabbit IgG Fc detection antibody, wash, substrate display solution, dilution/blocking solution, and stop solution.
8. The kit of any one of claims 4-6, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.
9. The kit of any one of claims 4-6, wherein the kit is a modified indirect enzyme-linked immunosorbent assay (ELISA) kit.
10. A method for quantitatively detecting the content of anti-human interferon alpha receptor 1 (IFNAR 1) monoclonal antibodies, wherein the antigen, primary antibody and secondary antibody referred to in claim 4 are used for ELISA detection using a modified indirect enzyme-linked immunosorbent assay.
11. The method of claim 10, comprising the steps of:
fixing the antigen to a solid phase carrier, and coating and sealing;
adding a first antibody, incubating to enable the first antibody to react with the coated antigen, and then adding a second antibody for incubation;
adding a diluted goat anti-rabbit IgG Fc detection antibody marked by horseradish peroxidase, and incubating;
adding a substrate color development solution, adding a stop solution after light-shielding incubation, measuring an OD value, and preparing a standard curve;
substituting the OD value of the measured sample into an equation to calculate the content of the anti-human IFNAR1 monoclonal antibody in the measured sample.
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| CN106243226A (en) * | 2016-08-05 | 2016-12-21 | 北京智仁美博生物科技有限公司 | Antibody of anti-human IFNAR1 and application thereof |
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