CN116675771A - Anti-human TSLP monoclonal antibody, kit containing same and inspection method - Google Patents
Anti-human TSLP monoclonal antibody, kit containing same and inspection method Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/53—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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- G—PHYSICS
- G01—MEASURING; TESTING
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- 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
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- 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]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/54—Interleukins [IL]
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Abstract
The present application provides an anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein: the amino acid sequence of CDR-H1 is shown in SEQ ID NO:1 or SEQ ID NO: 11; the amino acid sequence of CDR-H2 is shown in SEQ ID NO:2 or SEQ ID NO: shown at 12; the amino acid sequence of CDR-H3 is shown in SEQ ID NO:3 or SEQ ID NO: 13; the amino acid sequence of CDR-L1 is shown in SEQ ID NO:4 or SEQ ID NO: 14; the amino acid sequence of CDR-L2 is shown in SEQ ID NO:5 or SEQ ID NO: 15; the amino acid sequence of CDR-L3 is shown in SEQ ID NO:6 or SEQ ID NO: shown at 16. The application also provides an ELISA kit for detecting human TSLP and an inspection method. The kit provided by the application has the advantages of higher detection sensitivity, better detection specificity and good development and application prospects.
Description
Technical Field
The present application relates to the field of antibody pharmaceuticals. In particular, the application relates to monoclonal antibodies directed against human Thymic Stromal Lymphopoietin (TSLP), kits comprising the same, and methods of examination.
Background
Thymic Stromal Lymphopoietin (TSLP), a protein of the cytokine family, is produced primarily by non-hematopoietic cells, such as fibroblasts, epithelial cells, and different types of stromal or stromal-like cells. Typically expressed in epithelial cells at the surface of the lung, skin and intestinal barrier. The expression of TSLP in the respiratory tract of asthmatics is higher than normal and plays an important role in the maturation of T cell populations by activating antigen presenting cells. TSLP forms a ternary signaling complex with the thymic stromal lymphocyte receptor CRLF2 (TSLPR) and the IL-7Rα chain. Activation of intracellular signaling TSLP by activating STATs and JAK2 pathways then drives the release of T2 cytokines downstream including IL-4, IL-5 and IL-13, leading to inflammation and asthma symptoms. TSLP also activates multiple cell types that are involved in non-T2 driven inflammation.
The TSLP-TSLPR and downstream signal molecules are proved to be involved in the pathological processes of asthma and other various allergic diseases, and are potential therapeutic targets of allergic and autoimmune diseases, so that the development of TSLP-specific neutralizing antibodies has good application value for the prevention and treatment of related diseases.
The ELISA kit for measuring the TSLP content in the current market has the advantages of few types and high price, can quantitatively detect the TSLP content in a cell culture medium, is simple and convenient to operate, has high sensitivity and good specificity, and has important significance for researching pathogenesis of TSLP-related autoimmune diseases, a drug model and the like.
Disclosure of Invention
The application aims to provide an ELISA kit for detecting human TSLP, which is developed based on a modified double-antibody sandwich ELISA method and has high affinity for TSLP, and the kit comprises a pair of anti-human TSLP monoclonal antibodies. The kit can be used for realizing quantitative detection of human TSLP.
The technical scheme of the application is as follows:
1. an anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein:
the amino acid sequence of CDR-H1 is shown in SEQ ID NO:1 or SEQ ID NO: 11;
the amino acid sequence of CDR-H2 is shown in SEQ ID NO:2 or SEQ ID NO: shown at 12;
the amino acid sequence of CDR-H3 is shown in SEQ ID NO:3 or SEQ ID NO: 13;
the amino acid sequence of CDR-L1 is shown in SEQ ID NO:4 or SEQ ID NO: 14;
the amino acid sequence of CDR-L2 is shown in SEQ ID NO:5 or SEQ ID NO: 15;
the amino acid sequence of CDR-L3 is shown in SEQ ID NO:6 or SEQ ID NO: shown at 16.
2. The monoclonal antibody according to item 1, wherein the CDR-H1 has an amino acid sequence as shown in SEQ ID NO. 1, the CDR-H2 has an amino acid sequence as shown in SEQ ID NO. 2, the CDR-H3 has an amino acid sequence as shown in SEQ ID NO. 3, the CDR-L1 has an amino acid sequence as shown in SEQ ID NO. 4, the CDR-L2 has an amino acid sequence as shown in SEQ ID NO. 5, and the CDR-L3 has an amino acid sequence as shown in SEQ ID NO. 6.
3. The monoclonal antibody of item 1, wherein the CDR-H1 has an amino acid sequence as shown in SEQ ID NO. 11, the CDR-H2 has an amino acid sequence as shown in SEQ ID NO. 12, the CDR-H3 has an amino acid sequence as shown in SEQ ID NO. 13, the CDR-L1 has an amino acid sequence as shown in SEQ ID NO. 14, the CDR-L2 has an amino acid sequence as shown in SEQ ID NO. 15, and the CDR-L3 has an amino acid sequence as shown in SEQ ID NO. 16.
4. The monoclonal antibody of item 1 comprising a heavy chain variable region HCVR and a light chain variable region LCVR, wherein,
the amino acid sequence of the heavy chain variable region HCVR is shown in SEQ ID NO:7 or SEQ ID NO:17 or with SEQ ID NO:7 or SEQ ID NO:17, an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or more;
the amino acid sequence of the light chain variable region LCVR is shown in SEQ ID NO:8 or SEQ ID NO:18 or with SEQ ID NO:8 or SEQ ID NO:18 has an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical.
5. The monoclonal antibody of item 4, wherein the antibody heavy chain variable region HCVR has an amino acid sequence as set forth in SEQ ID No. 7 or an amino acid sequence identical to SEQ ID NO:7 having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical, and the antibody light chain variable region LCVR has an amino acid sequence as set forth in sequence SEQ ID No. 8 or an amino acid sequence that is identical to SEQ ID NO:8 has an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical.
6. The monoclonal antibody of item 4, wherein the antibody heavy chain variable region HCVR has an amino acid sequence as set forth in SEQ ID No. 17 or an amino acid sequence identical to SEQ ID NO:17 having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical, said antibody light chain variable region LCVR having an amino acid sequence as set forth in sequence SEQ ID NO:18 or an amino acid sequence identical to SEQ ID NO:18 has an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical.
7. The monoclonal antibody according to any one of items 1-6, comprising a heavy chain and a light chain, wherein:
the heavy chain has an amino acid sequence shown as SEQ ID NO. 9 or SEQ ID NO. 19 or an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity with SEQ ID NO. 9 or SEQ ID NO. 19;
the amino acid sequence of the light chain is shown as SEQ ID NO. 10 or SEQ ID NO. 20 or the amino acid sequence which has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% of identity with SEQ ID NO. 10 or SEQ ID NO. 20.
8. The monoclonal antibody according to item 7, which is an anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody 8D6 having a heavy chain with an amino acid sequence as shown in SEQ ID NO. 9 or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity to SEQ ID NO. 9, and a light chain with an amino acid sequence as shown in SEQ ID NO. 10 or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity to SEQ ID NO. 10.
9. The monoclonal antibody according to item 7, which is an anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody 4A5 having a heavy chain with an amino acid sequence as shown in SEQ ID NO. 19 or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity to SEQ ID NO. 19, and a light chain with an amino acid sequence as shown in SEQ ID NO. 20 or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity to SEQ ID NO. 20.
10. An ELISA kit to detect human TSLP, the ELISA kit comprising:
a first antibody (coated antibody) which has been immobilized to a solid support or is to be immobilized to the solid support; and
a biotin-labeled secondary antibody (detection antibody);
wherein the first antibody and the second antibody are monoclonal antibodies according to any one of items 1 to 9.
11. The ELISA kit of item 10, wherein the first antibody is anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody 8D6 and the second antibody is anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody 4A5.
12. The ELISA kit of item 10 or 11, further comprising peroxidase-labeled streptavidin, recombinant human TSLP protein standard, substrate, coated antibody diluent, wash solution, blocking solution/sample diluent, and stop solution.
13. The ELISA kit according to any of claims 10 to 12, which is a modified double antibody sandwich ELISA kit.
14. Use of an ELISA kit according to any of claims 10-13 to detect Thymic Stromal Lymphopoietin (TSLP) in cell culture broth, human serum.
15. A method for detecting Thymic Stromal Lymphopoietin (TSLP) content in a sample by ELISA using the antibody of any one of claims 1-9 using a modified double antibody sandwich method.
16. The method of item 15, comprising the step of,
coating an ELISA plate with an anti-human Thymus Stromal Lymphopoietin (TSLP) monoclonal antibody 8D 6;
adding a sample to be tested into the coated ELISA plate, and incubating;
adding a biotin-labeled anti-human Thymus Stromal Lymphopoietin (TSLP) monoclonal antibody 4A5 to an ELISA plate, and incubating;
diluting the peroxidase-labeled streptavidin, adding the diluted streptavidin into an ELISA plate, and incubating;
Adding a substrate into an ELISA plate, incubating in a dark place, adding a stop solution, and measuring an OD value;
and fitting a standard curve by using the OD value of the recombinant human TSLP protein standard substance, and substituting the OD value of the measured sample into an equation to calculate the content of Thymic Stromal Lymphopoietin (TSLP) in the measured sample.
The application provides an ELISA kit containing an anti-human TSLP monoclonal antibody. The kit is used for measuring the level of TSLP in a sample by using an improved double-antibody sandwich ELISA kit and carrying out Biotin labeling on an anti-human TSLP monoclonal antibody, so that a Biotin-avidin system (Biotin-AvidinSystem, BAS) can be formed by the kit and an enzyme-labeled secondary antibody, and the high-affinity firm combination of the kit can play a role in multilevel amplification of biological reaction, so that the kit has higher detection sensitivity, better detection specificity and good development and application prospects.
Drawings
The drawings are included to provide a better understanding of the application and are not to be construed as unduly limiting the application. Wherein:
FIG. 1 shows the binding of anti-human TSLP monoclonal antibody 8D6 (primary antibody) and anti-human TSLP monoclonal antibody 4A5 (secondary antibody) to human TSLP.
FIG. 2 shows the competitive inhibition of the anti-human TSLP monoclonal antibody 8D6 (primary antibody) and anti-human TSLP monoclonal antibody 4A5 (secondary antibody) with the antigen human TSLP binding site.
FIG. 3 is a standard graph of absorbance at OD450 for human TSLP.
FIG. 4 is a specific study of ELISA kits for detecting human TSLP.
Detailed Description
Exemplary embodiments of the application are described below, including various details of embodiments of the application to facilitate understanding, which should be considered as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the application. Also, descriptions of well-known functions and constructions are omitted in the following description for clarity and conciseness.
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 according to the application 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.
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 refers to an inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). 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, "anti-human TSLP monoclonal antibody" means such a monoclonal antibody: it is capable of binding human TSLP with sufficient affinity such that the monoclonal antibodies can be used to identify/detect human TSLP.
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 double antibody sandwich method is the most common method for detecting antigens. The antiserum containing known antibody is adsorbed in a small hole on a micro-titer plate and washed once; adding an antigen to be detected, if the antigen and the antigen are specific, combining, and then washing out redundant antibodies; adding an enzyme-linked antibody which specifically reacts with an antigen to be detected to form a sandwich; the addition of the substrate for the enzyme indicates the presence of the corresponding antigen if colored enzymatic products are observed.
In the present specification, "avidin" is a glycoprotein consisting of 4 subunits per molecule, and can be closely bound to 4 biotin molecules. More streptavidin (streptavidin) extracted from Streptomyces is used.
In the present specification, "biotin" is also called vitamin H. Chemically prepared derivatives, biotin-hydroxysuccinimide (BNCHS), can form biotinylated products with various types of large and small molecules such as proteins, carbohydrates and enzymes. The binding of avidin and biotin is not immune, but has strong specificity and high affinity, and once the avidin and biotin are combined, the binding is extremely stable. Since 1 avidin molecule has 4 binding sites for biotin molecules, more biotinylated molecules can be attached to form a lattice-like complex. Thus, combining avidin and biotin with ELISA can greatly increase the sensitivity of ELISA.
The biotin-avidin system is used in ELISA in various forms, and can be used for indirect coating and final reaction amplification. The enzyme-labeled antibody in conventional ELISA can also be replaced with biotinylated antibody, followed by ligation of avidin-enzyme conjugates to amplify the reaction signal.
In the present specification, human thymic stromal lymphopoietin (Human Thymic Stromal Lymphopoietin, TSLP) represents a pleiotropic cytokine derived from humans, the amino acid sequence of which is set forth in SEQ ID NO:21, wherein the underlined section represents the signal peptide.
SEQ ID NO：21：
MFPFALLYVLSVSFRKIFILQLVGLVLTYDFTNCDFEKIKAAYLSTISKDLI
TYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKT
KAALAIWCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ。
TSLP stimulates the production of cytokines by binding to its receptor, and is involved in the pathological processes of asthma and various allergic diseases, and the monoclonal antibody of the present application is a neutralizing antibody specifically recognizing TSLP, which is capable of specifically binding to TSLP.
The anti-human TSLP monoclonal antibodies of the present application do not bind to target-independent proteins. Herein, "unrelated proteins" refer to proteins other than human TSLP as a target; here, "not bonded" means: in the case where the binding capacity of the anti-human TSLP monoclonal antibody of the present application to human TSLP as its target is taken as 100%, the binding capacity of the anti-human TSLP monoclonal antibody of the present application to the unrelated protein is less than 10%, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.
The anti-human TSLP monoclonal antibodies of the present application may not bind TSLPs of other animal species. Here, "other animal species" refers to other animal species than humans, such as marmoset, cynomolgus monkey, pig, dog, rabbit, rat, mouse, guinea pig, and the like; here, "not bonded" means: in the case where the binding capacity of the anti-human TSLP monoclonal antibody of the present application to human TSLP as its target is taken as 100%, the binding capacity of the anti-human TSLP monoclonal antibody of the present application to TSLPs of other animal species is less than 10%, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.
The anti-human TSLP monoclonal antibodies of the present application have equilibrium dissociation constants (K) of 1. Mu.M, 100nM, 50nM, 40nM or less D )。
Experimental results show that the anti-human TSLP monoclonal antibodies of the present application can specifically bind to human TSLP.
The anti-human TSLP monoclonal antibody of the application is equivalent to or superior to the same monoclonal antibody product on the market in terms of various biological activities. Such as neutralizing the activity of STAT5 phosphorylation in human, natural, cynomolgus TSLP-induced cells, neutralizing the activity of human TSLP-induced human whole blood, human PBMC cells to release TARC (CCL 17), and the like.
In this specification "identity" refers to the relationship between sequences of two or more polypeptides (e.g. antigens) or polynucleotides (nucleic acids) as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between sequences as determined by the number of matches between two or more amino acid residues or strings of nucleic acid residues. Identity measures the percentage of identical matches between smaller sequences in two or more sequences, with gap alignments (if present) addressed by a specific mathematical model or computer program (e.g. "algorithm"). Identity of the relevant antigen or nucleic acid can be readily calculated by known methods. "percent identity (%)" when applied to a polypeptide or polynucleotide sequence is defined as the percentage of residues in a candidate amino acid or nucleotide sequence that are identical to residues in the amino acid sequence or nucleotide sequence of the second sequence (amino acid residues or nucleic acid residues) after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art. It will be appreciated that identity depends on the calculation of percent identity, but its value may vary due to gaps and penalties introduced in the calculation.
In the present application, the anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibodies are two, one is the first antibody 8D6 and the other is the second antibody 4A5.
Specifically, the anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody 8D6, comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein:
the amino acid sequence of CDR-H1 is shown in SEQ ID NO:1 (GYVMG);
the amino acid sequence of CDR-H2 is shown in SEQ ID NO:2 (SIGVDGDTVYATWPKG);
the amino acid sequence of CDR-H3 is shown in SEQ ID NO:3 (RYDGRNVVPGTFDV);
the amino acid sequence of CDR-L1 is shown in SEQ ID NO:4 (QASESISKWLA);
the amino acid sequence of CDR-L2 is shown in SEQ ID NO:5 (SASDLAS);
the amino acid sequence of CDR-L3 is shown in SEQ ID NO:6 (QQDYTDYGVDNP).
Further, the monoclonal antibody 8D6 comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein,
further, the heavy chain variable region HCVR has an amino acid sequence as set forth in SEQ ID NO:7 (QSVEESGGGLVTPGTPLTLTCTVSGFSLSGYVMGWVRQAPGKGLEWI GSIGVDGDTVYATWPKGRFTISKTSTTVDLKITSPTTEDTATYFCVRRYD GRNVVPGTFDVWGPGTLVTVSS);
Further, the heavy chain variable region HCVR has an amino acid sequence as set forth in SEQ ID NO:7 has an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical;
further, the amino acid sequence of the light chain variable region LCVR is as set forth in SEQ ID NO:8 (AYDMTQTPASVEVAVGGTVTINCQASESISKWLAWYQQKPGQPPKLLI YSASDLASGVPSRFKGSGSGTQFTLTISGVECDDAATYYCQQDYTDYGVD NPFGGGTEVVVK).
Further, the amino acid sequence of the light chain variable region LCVR is as set forth in SEQ ID NO:8 has an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical;
further, the monoclonal antibody 8D6 comprises a heavy chain and a light chain, wherein:
the amino acid sequence of the heavy chain is shown as SEQ ID NO. 9 (QSVEESGGGLVTPGTPLTLTCTVSGFSLSGYVMGWVRQAPGKGLEWI GSIGVDGDTVYATWPKGRFTISKTSTTVDLKITSPTTEDTATYFCVRRYDGRNVVPGTFDVWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK);
The amino acid sequence of the heavy chain has the amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% of the identity with SEQ ID No. 9;
the amino acid sequence of the light chain is shown as SEQ ID NO. 10 (AYDMTQTPASVEVAVGGTVTINCQASESISKWLAWYQQKPGQPPKLLI YSASDLASGVPSRFKGSGSGTQFTLTISGVECDDAATYYCQQDYTDYGVDNPFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC).
The amino acid sequence of the light chain is as shown as the amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% of the identity with SEQ ID NO. 10.
Specifically, the anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody 4A5, comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein:
the amino acid sequence of CDR-H1 is shown in SEQ ID NO:11 (SYWMT) shown;
the amino acid sequence of CDR-H2 is shown in SEQ ID NO:12 (IISSSGASYYASWAKG);
the amino acid sequence of CDR-H3 is shown in SEQ ID NO:13 (REFVSGYYIFKL);
the amino acid sequence of CDR-L1 is shown in SEQ ID NO:14 (QASLSISTSLA);
The amino acid sequence of CDR-L2 is shown in SEQ ID NO:15 (GASNLAS) as shown;
the amino acid sequence of CDR-L3 is shown in SEQ ID NO:16 (QSNYDSTNYA).
Further, the monoclonal antibody 4A5 comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein,
the amino acid sequence of the heavy chain variable region HCVR is shown in SEQ ID NO:17 (QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYWMTWVRQAPGKGLEWIGI ISSSGASYYASWAKGRFTISKTSSTTVDLRITSPTTEDTATYFCGRREFVSG YYIFKLWGQGTLVTVSS);
the amino acid sequence of the heavy chain variable region HCVR is as shown in SEQ ID NO:17, an amino acid sequence having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
The amino acid sequence of the light chain variable region LCVR is shown in SEQ ID NO:18 (AELLMTQTPASVSEPVGGTVTIKCQASLSISTSLAWYQQKPGQPPKLLI YGASNLASGVSSRFKGSRSGTQFTLTISDLECADAATYHCQSNYDSTNYA FGGGTEVVVK).
The amino acid sequence of the light chain variable region LCVR is as shown in SEQ ID NO:18 has an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical.
Further, the monoclonal antibody 4A5 comprises a heavy chain and a light chain, wherein:
The amino acid sequence of the heavy chain is shown as SEQ ID NO. 19 (QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYWMTWVRQAPGKGLEWIG IISSSGASYYASWAKGRFTISKTSSTTVDLRITSPTTEDTATYFCGRREFVSGYYIFKLWGQGTLVTVSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK);
the amino acid sequence of the heavy chain is as shown as the amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% of the identity with SEQ ID NO. 19.
The amino acid sequence of the light chain is shown as SEQ ID NO. 20 (AELLMTQTPASVSEPVGGTVTIKCQASLSISTSLAWYQQKPGQPPKLLI YGASNLASGVSSRFKGSRSGTQFTLTISDLECADAATYHCQSNYDSTNYAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC).
The amino acid sequence of the light chain is as shown as the amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% of the identity with SEQ ID NO. 20.
The application also provides an ELISA kit for detecting human TSLP, which comprises:
a first antibody (coated antibody) which has been immobilized to a solid support or is to be immobilized to the solid support; and
a biotin-labeled secondary antibody (detection antibody);
the first antibody is the aforementioned anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody 8D6, and the second antibody is the aforementioned anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody 4A5.
Specifically, the ELISA kit further comprises peroxidase-labeled streptavidin, recombinant human TSLP protein standard, substrate, coated antibody diluent, washing liquid, blocking liquid/sample diluent and stop liquid.
Specifically, the solid phase carrier is an ELISA plate. Preferably 96-well ELISA plates, in particular polystyrene plastic plates of Corning Coster, U.S.A. The biotin is as follows: biotin. The peroxidase is horseradish peroxidase (HRP), and the labeled streptavidin is streptavidin-horseradish peroxidase conjugate (SA-HRP). The substrate is 3,3', 5' -tetramethyl benzidine (TMB); the coated antibody diluent is Phosphate Buffer (PBS) pH7.4; the wash solution was Phosphate Buffered Saline (PBS) containing 0.05% Tween20; blocking/sample dilutions were Phosphate Buffered Saline (PBS) containing 0.5% bsa, 0.05% tween20 and 0.05% proclin300; the stop solution is phosphoric acid.
Specifically, the ELISA kit comprises:
1) Solid phase carrier: the ELISA plate is 1 block;
2) Coating an antibody: primary antibody 8D6, 10 μg/tube, 1 tube;
3) Detection of antibodies: second antibody 4A5 (labeled with biotin), 10 μg/tube, 1 tube;
4) Standard substance: recombinant human TSLP protein, 10 ng/tube, 1 tube;
5) Peroxidase-labeled streptavidin: streptavidin-horseradish peroxidase conjugate (strepavidin-HRP, SA-HRP for short), 500 ng/tube, 1 tube;
6) A substrate: 3,3', 5' -tetramethyl benzidine (TMB), 5 ml/tube, 1 tube each for liquid A and liquid B;
7) Coating antibody diluent: phosphate Buffered Saline (PBS), pH 7.4, 50 ml/bottle, 1 bottle;
8) Washing liquid: phosphate Buffered Saline (PBS) containing 0.05% Tween20, 200 ml/bottle, 1 bottle;
9) Blocking/sample dilution: phosphate Buffered Saline (PBS) containing 0.5% BSA, 0.05% Tween20 and 0.05% Proclin300, 200 ml/bottle, 1 bottle;
10 Termination liquid: 1M phosphoric acid, 10 ml/tube, 1 tube.
In a specific embodiment, the kit is an enzyme-linked immunosorbent assay (ELISA) kit. Preferably, the kit is a modified double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit.
The ELISA kit provided by the application is used for detecting Thymic Stromal Lymphopoietin (TSLP) in cell culture solution and human serum.
The application provides a method for detecting Thymic Stromal Lymphopoietin (TSLP) content in a sample, which uses the antibodies and utilizes a modified double antibody sandwich method to carry out ELISA detection.
In particular, the method comprises the steps of,
step one: coating an ELISA plate with an anti-human Thymus Stromal Lymphopoietin (TSLP) monoclonal antibody 8D 6;
step two: adding a sample to be tested into the coated ELISA plate, and incubating;
step three: adding a biotin-labeled anti-human Thymus Stromal Lymphopoietin (TSLP) monoclonal antibody 4A5 to an ELISA plate, and incubating;
step four: diluting the peroxidase-labeled streptavidin, adding the diluted streptavidin into an ELISA plate, and incubating;
step five: adding a substrate into an ELISA plate, incubating in a dark place, adding a stop solution, and measuring an OD value;
step six: and fitting a standard curve by using the OD value of the recombinant human TSLP protein standard substance, and substituting the OD value of the measured sample into an equation to calculate the content of Thymic Stromal Lymphopoietin (TSLP) in the measured sample.
In one embodiment, the ELISA kit of the application can be used for quantitatively detecting the content of human TSLP in a sample, and comprises the following steps:
(1) Coating: preparing a coated antibody 8D6 (primary antibody) into a coated antibody working solution with the concentration of 1 mu g/ml by adopting a coated antibody diluent, adding the coated antibody working solution into an ELISA plate (96-hole Coster ELISA plate) according to the dosage of 50 mu l/hole, and standing at the temperature of 2-8 ℃ overnight;
(2) Closing: the coated antibody working solution in the ELISA plate is discarded, the plate is washed 3 times by washing solution (phosphate buffer solution (PBS) containing 0.05% Tween 20), then a blocking solution (phosphate buffer solution (PBS) containing 0.5% BSA, 0.05% Tween20 and 0.05% proclin 300) is added according to the dosage of 100 mu l/hole, and the plate is blocked for 2 hours at room temperature in a shaking table (120 rpm);
(3) Protein standard preparation: 10 EP tubes were taken and numbered sequentially, 140 μl of sample diluent was added per tube from tube 2 and placed on the EP tube rack; preparing standard protein into liquid with the concentration of 4ng/ml by using a sample diluent, sucking 300 mu l of the liquid into a 1 st EP pipe, sucking 140 mu l of the liquid from the 1 st EP pipe, adding the liquid into a 2 nd EP pipe for double dilution, and the like to a 9 th EP pipe (the concentration of the last pipe is 0);
(4) Preparing a quality control sample: taking 5 EP tubes, sampling from 4ng/ml respectively, and preparing quality control samples of 1.6ng/ml, 0.8ng/ml, 0.4ng/ml, 0.2ng/ml and 0.05 ng/ml;
(5) Sample adding: removing the sealing liquid in the ELISA plate, washing the plate with washing liquid for 3 times, adding recombinant human TSLP protein standard liquid and quality control sample according to the dosage of 50 μl/hole, and placing in a shaking table (120 rpm) for incubation for 2 hours at room temperature;
(6) Adding a detection antibody: discarding a protein standard solution and a sample to be detected in the ELISA plate, washing the plate for 3 times by using a washing liquid, preparing a detection antibody anti-human TSLP monoclonal antibody (biotin labeling) into a detection antibody working solution with the concentration of 0.2 mug/ml by using a sample diluent, adding the detection antibody working solution into the ELISA plate according to the dosage of 50 mug/hole, and placing the ELISA plate in a shaking table (120 rpm) for incubation reaction for 2 hours;
(7) Adding enzyme-labeled secondary antibodies: discarding the detection antibody working solution in the ELISA plate, washing the plate for 3 times by using a washing liquid, preparing an ELISA secondary antibody (streptavidin-horseradish peroxidase conjugate (SA-HRP)) into an ELISA secondary antibody working solution with the concentration of 100ng/ml by using a sample diluent, adding the ELISA plate according to the dosage of 50 μl/hole, and placing the ELISA plate in a shaking table (120 rpm) for incubation for 1 hour at room temperature;
(8) 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) in equal volume; discarding the enzyme-labeled secondary antibody working solution in the enzyme-labeled plate, washing the plate for 3 times by using washing liquid, adding a substrate according to the dosage of 50 mu l/hole, and carrying out light-shielding reaction for 5-10 minutes at room temperature;
(9) Terminating the color development: and adding a termination solution into the ELISA plate according to the dosage of 50 mu l/hole to terminate the reaction, measuring the OD value of each hole in the ELISA plate by using an ELISA reader at the wavelength of 450nm, 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
The experimental methods used in the following examples are conventional methods, if no special requirements are imposed.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 preparation of monoclonal antibodies 8D6 (primary antibody) and 4A5 (secondary antibody)
The primary antibody was 8D6, the secondary antibody was 4A5, and both the primary antibody and the secondary antibody obtained in this embodiment were rabbit monoclonal antibodies, and were not humanized.
The preparation method comprises the following steps: the correct sequence of the first antibody and the second antibody heavy chain expression plasmid and the light chain expression plasmid respectively were co-transfected with the ExpiCHO-S cells. The day before transfection, the ExpiCHO-S cells were diluted to 3X 10 6 Each cell/ml was passaged before transfection. On the day of transfection, cell densities were diluted to 6X 10 6 25ml of cell suspension were shake-bottled at each cell/ml, 125ml, and waiting for transfection. Mu.l of OptiPRO SFM (available from Gibco) was added to wells 1 and 2, respectively, followed by 12.5. Mu.g of heavy and light chain plasmids in well 1 and 80. Mu. l ExpiFectamine CHO reagent (available from Gibco) in well 2. Transfer well 2 into well 1Mixing, standing for 1 min, and adding into cells. On the first day after transfection, 150. Mu.l of enhancement (from Gibco) and 6ml of Feed broth (from Gibco) were added. The sixth day after transfection, the culture supernatant was harvested and treated with Protein A @A, purchased from Merck) was purified in one step, and primary and secondary antibodies were obtained.
The complete sequences of the heavy and light chains of the primary and secondary antibodies (the complete sequences of the heavy and light chains of the primary antibody are shown in SEQ ID NO:9, SEQ ID NO:10, and the complete sequences of the heavy and light chains of the secondary antibody are shown in SEQ ID NO:19, and SEQ ID NO: 20) were obtained by sequencing (entrusting organisms), and then the CDR sequences thereof were obtained according to the Kabat rules (reference: kabat E.A., wu T.T., bilofsky H.Attempts to locate residues in complementarity-determining regions of antibody combining sites that make contact with anti.Proc.Natl.Acad.Sci.USA.1976; 73:617-619).
EXAMPLE 2 characterization of primary and secondary antibodies
1. Binding experiments of primary and secondary antibodies to human TSLP:
the elisa plate (from Thermo) was coated with primary and secondary antibodies, respectively, and after blocking, a gradient of human TSLP (His-tag, from ACRO Biosystems) was added and incubated for 2 hours at room temperature. After washing the plates, biotin-labeled anti-His tag antibodies were added and incubated for 1 hour at room temperature. After washing the plates, the detection antibody strepitavidin-HRP was added and incubated for 1 hour at room temperature. Finally, the plate is washed, developed and the absorbance at 450nm is read. The results are shown in FIG. 1, where both the primary and secondary antibodies specifically bind to human TSLP.
2. Competitive inhibition experiments of antibody recognition antigen sites:
the ELISA plate is coated with the primary antibody, the temperature is 2-8 ℃ overnight, and the next day is closed for 2 hours at room temperature. The primary antibody and secondary antibody were mixed with equal volumes of human TSLP at concentrations and incubated for 2 hours at room temperature. After washing the plates, incubated antibody and human TSLP complex were added and incubated for 2h at room temperature. After washing the plates, biotin-labeled anti-His tag antibodies were added and incubated for 1 hour at room temperature. After washing the plates, the detection antibody strepitavidin-HRP was added and incubated for 1 hour at room temperature. Finally, the plate is washed, developed and the absorbance at 450nm is read. As a result, as shown in FIG. 2, the primary antibody and the secondary antibody did not compete with the binding site of human TSLP antigen, and the secondary antibody was used as a detection antibody for human TSLP ELISA kit.
3. Detection antibody (secondary antibody) biotin label:
the detection antibody was labeled with Biotin using the sulfoNHS LC-Biotin kit (available from Thermo), and the specific procedure was strictly followed according to the kit instructions. After the labeling was completed, the free biotin was removed by filtration using a ZebaTM rotary desalting column (Spin Desalting Colum), and the resulting solution was a biotin-labeled detection antibody (secondary antibody).
Example 3 quantitative detection kit for human TSLP ELISA
The composition, specification, source, storage conditions and other information of the human TSLP ELISA quantitative determination kit are shown in Table 1 below.
TABLE 1 composition of ELISA kit components
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Example 4 quantitative detection of human TSLP Using the human TSLP ELISA kit of example 3
1. Sample treatment:
the recombinant human TSLP protein standard was diluted with a sample diluent, and a standard curve was fitted with the absorbance of the recombinant human TSLP protein standard diluted with a cell culture solution (90% RPMI1640 (from Hyclone) +10% FBS (from Hyclone)) as a sample to be examined. Substituting the OD value of the sample to be detected into the standard curve to obtain the theoretical concentration value of human TSLP in the sample to be detected, and then calculating the recovery rate (recovery rate=theoretical concentration value/actual concentration value×100) of the sample to be detected, thereby analyzing the feasibility of the ELISA kit in example 3 for quantitative detection of human TSLP in the cell culture fluid.
2. Quantitative detection operation steps:
(1) Coating: preparing coated antibody into coated antibody working solution with concentration of 1 mu g/ml by adopting coated antibody diluent, adding the coated antibody working solution into an ELISA plate according to the dosage of 50 mu l/hole, and standing at 4 ℃ overnight;
(2) Closing: discarding the coated antibody working solution in the ELISA plate, washing the plate with washing solution for 3 times, adding a sealing solution according to the dosage of 100 μl/hole, and sealing for 2 hours at room temperature in a shaking table (120 rpm);
(3) Recombinant human TSLP protein standard preparation: 10 EP tubes were taken and numbered sequentially, 140 μl of sample diluent was added per tube from tube 2 and placed on the EP tube rack; preparing standard protein into liquid with the concentration of 4ng/ml by using a sample diluent, sucking 300 mu l of the liquid into a 1 st EP pipe, sucking 140 mu l of the liquid from the 1 st EP pipe, adding the liquid into a 2 nd EP pipe for double dilution, and the like to a 9 th EP pipe (the concentration of the last pipe is 0);
(4) Adding a sample to be tested and a standard substance: removing the sealing liquid in the ELISA plate, washing the plate with washing liquid for 3 times, adding protein standard liquid and a sample to be tested according to the dosage of 50 mu l/hole, and placing the mixture in a shaking table (120 rpm) for incubation for 2 hours at room temperature;
(5) Adding a detection antibody: removing protein standard solution and sample to be detected in the ELISA plate, washing the plate for 3 times by using washing liquid, preparing detection antibody (biotin labeling) into detection antibody working solution with the concentration of 0.2 mu g/ml by using sample diluent, adding the detection antibody working solution into the ELISA plate according to the dosage of 50 mu l/hole, and placing the ELISA plate in a shaking table (120 rpm) for incubation reaction for 2 hours;
(6) Adding enzyme-labeled secondary antibodies: discarding the detection antibody working solution in the ELISA plate, washing the plate for 3 times by using a washing liquid, preparing the ELISA secondary antibody into an ELISA secondary antibody working solution with the concentration of 100ng/ml by using a sample diluent, adding the ELISA secondary antibody working solution into the ELISA plate according to the dosage of 50 μl/hole, and placing the ELISA plate in a shaking table (120 rpm) for incubation for 1 hour at room temperature;
(7) Color development: mixing the substrate A and B in equal volume; discarding the enzyme-labeled secondary antibody working solution in the enzyme-labeled plate, washing the plate for 3 times by using washing liquid, adding a substrate according to the dosage of 50 mu l/hole, and carrying out light-shielding reaction for 10-15 minutes at room temperature;
(8) Terminating the color development: and adding a termination solution into the ELISA plate according to the dosage of 50 mu l/hole to terminate the reaction, measuring the OD value of each hole in the ELISA plate by using an ELISA apparatus at the wavelength of 450nm, fitting a standard curve according to the OD value of a standard substance, substituting the OD value of a sample to be detected into an equation, and calculating the concentration of human TSLP in the sample to be detected, thereby finishing quantitative detection.
3. Detection result:
(1) The assay data for recombinant human TSLP standards are shown in Table 2 below:
TABLE 2
(2) Curve equation: 4-P Fit: y= (0.049-2.290)/(1+ (x/0.388)/(1.191) +2.290, r2=0.999 (see fig. 3).
(3) The test data of the samples to be tested diluted with the cell culture fluid are shown in the following table 3:
TABLE 3 Table 3
The recovery rate of each concentration point is 88% -109%, which shows that the cell culture solution has no influence on the experimental result, and the ELISA method can be suitable for the content measurement of human TSLP in the cell culture solution system.
4. Accuracy and precision investigation:
(1) In 3 different experiments, five human TSLPs (1.6, 0.8, 0.4, 0.2 and 0.05 ng/ml) with known concentrations are diluted by cell culture fluid to serve as samples to be tested, 2 compound holes are respectively arranged, quantitative detection of the human TSLPs is carried out, and the accuracy and precision of the kit are analyzed. Precision of the method is expressed as coefficient of variation, CV% = SD/average x 100. Accuracy is expressed as relative error re%, re% = (average observed concentration-nominal concentration)/nominal concentration x 100. The accuracy of the detection of the kit is analyzed through the parameters. The analysis results are shown in Table 4.
Table 4 quantitative detection accuracy and precision experimental data for human TSLP
(2) The results and discussion are as follows:
the results in Table 4 show that the coefficient of variation CV%. Ltoreq. 9.820% in batches at 5 concentration levels, with the relative error RE% in batches at 5 concentration levels ranging between-0.958% and 11.000%. The results show that the human TSLP for quantitatively detecting the sample by using the human TSLP ELISA kit provided by the application can meet the requirement that the variation coefficient and the relative error in the batch are less than or equal to 20%, and the detection method provided by the application has good accuracy.
5. Specificity investigation:
different concentration gradients of human IL-4, human IL-13, human IL-5, human IL-7 and human TSLP proteins (4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0 ng/ml) were added to the detection wells of the primary antibody pre-coated ELISA kit, and absorbance of each protein at different concentrations was determined using the ELISA assay established in example 4. As a result, as shown in FIG. 4, the OD450 of the human TSLP protein group exhibited a significant dose-dependent effect between the different concentrations, the OD450 absorbance increased with increasing protein concentration, whereas the absorbance values of the human IL-4, human IL-13, human IL-5, and human IL-7 protein groups were all close to 0, and there was no dose-dependent effect. The experimental results show that the detection method using the human TSLP ELISA kit of example 3 has good specificity.
Although the embodiments of the present application have been described above, the present application is not limited to the above-described specific embodiments and application fields, and the above-described specific embodiments are merely illustrative, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous forms of the application without departing from the scope of the application as claimed.
Claims (16)
1. An anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein:
the amino acid sequence of CDR-H1 is shown in SEQ ID NO:1 or SEQ ID NO: 11;
the amino acid sequence of CDR-H2 is shown in SEQ ID NO:2 or SEQ ID NO: shown at 12;
the amino acid sequence of CDR-H3 is shown in SEQ ID NO:3 or SEQ ID NO: 13;
the amino acid sequence of CDR-L1 is shown in SEQ ID NO:4 or SEQ ID NO: 14;
the amino acid sequence of CDR-L2 is shown in SEQ ID NO:5 or SEQ ID NO: 15;
the amino acid sequence of CDR-L3 is shown in SEQ ID NO:6 or SEQ ID NO: shown at 16.
2. The monoclonal antibody of claim 1, wherein the CDR-H1 has an amino acid sequence as shown in SEQ ID No. 1, the CDR-H2 has an amino acid sequence as shown in SEQ ID No. 2, the CDR-H3 has an amino acid sequence as shown in SEQ ID No. 3, the CDR-L1 has an amino acid sequence as shown in SEQ ID No. 4, the CDR-L2 has an amino acid sequence as shown in SEQ ID No. 5, and the CDR-L3 has an amino acid sequence as shown in SEQ ID No. 6.
3. The monoclonal antibody of claim 1, wherein the CDR-H1 has an amino acid sequence as shown in SEQ ID No. 11, the CDR-H2 has an amino acid sequence as shown in SEQ ID No. 12, the CDR-H3 has an amino acid sequence as shown in SEQ ID No. 13, the CDR-L1 has an amino acid sequence as shown in SEQ ID No. 14, the CDR-L2 has an amino acid sequence as shown in SEQ ID No. 15, and the CDR-L3 has an amino acid sequence as shown in SEQ ID No. 16.
4. The monoclonal antibody of claim 1, wherein the monoclonal antibody comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein,
the amino acid sequence of the heavy chain variable region HCVR is shown in SEQ ID NO:7 or SEQ ID NO:17 or with SEQ ID NO:7 or SEQ ID NO:17, an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or more;
the amino acid sequence of the light chain variable region LCVR is shown in SEQ ID NO:8 or SEQ ID NO:18 or with SEQ ID NO:8 or SEQ ID NO:18 has an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical.
5. The monoclonal antibody of claim 4, wherein the antibody heavy chain variable region HCVR has an amino acid sequence as set forth in SEQ ID No. 7 or a sequence identical to SEQ ID NO:7 having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical, and the antibody light chain variable region LCVR has an amino acid sequence as set forth in SEQ ID No. 8 or an amino acid sequence identical to SEQ ID NO:8 has an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical.
6. The monoclonal antibody of claim 4, wherein the antibody heavy chain variable region HCVR has an amino acid sequence as set forth in SEQ ID No. 17 or a sequence identical to SEQ ID NO:17 having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical, said antibody light chain variable region LCVR having an amino acid sequence as set forth in sequence SEQ ID NO:18 or an amino acid sequence identical to SEQ ID NO:18 has an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical.
7. The monoclonal antibody of any one of claims 1-6, wherein the monoclonal antibody comprises a heavy chain and a light chain, wherein:
the heavy chain has an amino acid sequence shown as SEQ ID NO. 9 or SEQ ID NO. 19 or an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity with SEQ ID NO. 9 or SEQ ID NO. 19;
the amino acid sequence of the light chain is shown as SEQ ID NO. 10 or SEQ ID NO. 20 or the amino acid sequence which has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% of identity with SEQ ID NO. 10 or SEQ ID NO. 20.
8. The monoclonal antibody of claim 7, wherein the monoclonal antibody is an anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody 8D6, the heavy chain of which has the amino acid sequence shown in SEQ ID No. 9 or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity to SEQ ID No. 9, and the light chain has the amino acid sequence shown in SEQ ID No. 10 or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity to SEQ ID No. 10.
9. The monoclonal antibody of claim 7, wherein the monoclonal antibody is anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody 4A5, the heavy chain of which has the amino acid sequence shown in SEQ ID No. 19 or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity to SEQ ID No. 19, and the light chain has the amino acid sequence shown in SEQ ID No. 20 or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity to SEQ ID No. 20.
10. An ELISA kit to detect human TSLP, wherein the ELISA kit comprises:
a first antibody (coated antibody) which has been immobilized to a solid support or is to be immobilized to the solid support; and
a biotin-labeled secondary antibody (detection antibody);
wherein the first antibody and the second antibody are monoclonal antibodies according to any one of claims 1 to 9.
11. The ELISA kit according to claim 10, wherein the first antibody is an anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody 8D6 and the second antibody is an anti-human Thymic Stromal Lymphopoietin (TSLP) monoclonal antibody 4A5.
12. The ELISA kit according to claim 10 or 11, wherein the ELISA kit further comprises peroxidase-labeled streptavidin, recombinant human TSLP protein standard, substrate, coated antibody dilution, wash solution, blocking/sample dilution, and stop solution.
13. The ELISA kit according to any of claims 10-12, wherein the kit is a modified double antibody sandwich ELISA kit.
14. Use of an ELISA kit according to any of claims 10-13 for the detection of Thymic Stromal Lymphopoietin (TSLP) in cell culture broth, human serum.
15. A method for detecting Thymic Stromal Lymphopoietin (TSLP) content in a sample, wherein an ELISA assay is performed using the antibody of any one of claims 1-9 using a modified double antibody sandwich method.
16. The method of claim 15, comprising the step of,
coating an ELISA plate with an anti-human Thymus Stromal Lymphopoietin (TSLP) monoclonal antibody 8D 6;
adding a sample to be tested into the coated ELISA plate, and incubating;
adding a biotin-labeled anti-human Thymus Stromal Lymphopoietin (TSLP) monoclonal antibody 4A5 to an ELISA plate, and incubating;
diluting the peroxidase-labeled streptavidin, adding the diluted streptavidin into an ELISA plate, and incubating;
adding a substrate into an ELISA plate, incubating in a dark place, adding a stop solution, and measuring an OD value;
and fitting a standard curve by using the OD value of the recombinant human TSLP protein standard substance, and substituting the OD value of the measured sample into an equation to calculate the content of Thymic Stromal Lymphopoietin (TSLP) in the measured sample.
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