CN114195892A - Detection control method for human insulin single-chain precursor residue - Google Patents
Detection control method for human insulin single-chain precursor residue Download PDFInfo
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- CN114195892A CN114195892A CN202010990334.3A CN202010990334A CN114195892A CN 114195892 A CN114195892 A CN 114195892A CN 202010990334 A CN202010990334 A CN 202010990334A CN 114195892 A CN114195892 A CN 114195892A
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- human insulin
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- chain precursor
- monoclonal antibody
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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Abstract
The invention relates to a detection control method for human insulin single-chain precursor residue. Specifically, the invention relates to an anti-human insulin single-chain precursor monoclonal antibody, which specifically recognizes and binds to a protein shown in SEQ ID NO. 1. The monoclonal antibody of the human insulin single-chain precursor can be used as a coating antibody or a detection antibody in an enzyme-linked immunosorbent assay method and is used for quickly and accurately detecting the human insulin single-chain precursor.
Description
Technical Field
The invention belongs to the analysis and detection technology in the field of biological pharmacy, and particularly relates to a detection control method for human insulin single-chain precursor residue.
Background
Diabetes is a syndrome of abnormal metabolism of sugars, proteins, fats and aqueous mediators due to insufficient insulin secretion and reduced sensitivity of target cells to insulin for a variety of reasons. Insulin is a protein hormone secreted by beta cells stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon and the like, is the only hormone for reducing blood sugar in the body, and is the only hormone for simultaneously promoting glycogen, fat and protein synthesis.
Insulin is an epoch-making hypoglycemic drug, and since the approval of human insulin on the market in 1982, insulin has been widely used in clinical treatment of type II diabetes. Human insulin is a protein consisting of 51 amino acid residues produced by recombinant DNA technology, and the host cell is Escherichia coli. The content of human insulin per 1mg of dry product should not be less than 27.5 units.
The production modes of human insulin are mainly divided into 2 types: firstly, A, B chains of human insulin are respectively produced by fermentation of genetically engineered escherichia coli, and then a disulfide bond is formed on A, B chains under certain conditions through a chemical oxidation method, so that the human insulin is obtained; secondly, the insulin precursor is produced by fermentation of genetic engineering escherichia coli or yeast, and active insulin is formed by the steps of enzyme digestion, purification and the like. The second method is now the most commonly used means for producing insulin.
However, at present, no specific detection method is specified for the detection of the human insulin single-chain precursor in the Chinese pharmacopoeia, European pharmacopoeia and United states pharmacopoeia, and each production enterprise adopts a proper method for detection, so that the liquid phase method has poor separation effect and sensitivity for the process products and the raw material medicines in the production process of the enterprise. Therefore, the method for detecting the single-chain precursor residue, which has the advantages of high sensitivity, strong specificity and good repeatability, is provided, so that the single-chain precursor residue in the process product and the raw material medicine in the process is accurately detected, and the safety of the biological product and the quality control in the production process are guaranteed.
Therefore, there is a need to develop an efficient method for the detection of human insulin precursor.
Disclosure of Invention
The invention aims to provide an anti-human insulin single-chain precursor monoclonal antibody which can be used as a coating antibody or a detection antibody in an enzyme-linked immunosorbent assay (ELISA) method and is used for quickly and accurately detecting a human insulin single-chain precursor.
The invention provides an anti-human insulin single-chain precursor monoclonal antibody, which specifically recognizes and combines with a protein shown in SEQ ID NO. 1.
In another preferred embodiment, the anti-human insulin single-chain precursor is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156.
In the second aspect of the invention, a hybridoma cell strain is provided, wherein the hybridoma cell strain is used for producing the anti-human insulin single-chain precursor monoclonal antibody in the first aspect of the invention, and the preservation number of the hybridoma cell strain is CCTCC NO: C2020138 or CCTCC NO: C2020156.
In a third aspect of the present invention, there is provided a recombinant protein having:
(i) an anti-human insulin single chain precursor monoclonal antibody according to the first aspect of the invention; and
(ii) optionally a tag sequence to facilitate expression and/or purification.
In a fourth aspect of the invention, there is provided an immunoconjugate comprising:
(a) an anti-human insulin single chain precursor monoclonal antibody according to the first aspect of the invention; and
(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, and the like.
In a fifth aspect of the invention there is provided a polynucleotide encoding an anti-human insulin single chain precursor monoclonal antibody according to the first aspect of the invention or a recombinant protein according to the third aspect of the invention.
In a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) an anti-human insulin single chain precursor monoclonal antibody according to the first aspect of the invention, or a recombinant protein according to the third aspect of the invention, or an immunoconjugate according to the fourth aspect of the invention; and
(ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is in the form of injection.
In a seventh aspect of the invention, there is provided the use of an anti-human insulin single chain precursor monoclonal antibody according to the first aspect of the invention, or a recombinant protein according to the third aspect of the invention, or an immunoconjugate according to the fourth aspect of the invention, for the manufacture of a medicament, a reagent, a test plate or a kit;
the reagent, assay plate or kit is for:
(1) the protein shown as SEQ ID NO.1 in the detected sample.
In another preferred embodiment, the sample comprises a human insulin single chain precursor.
In another preferred embodiment, the sample contains a human insulin single-chain precursor and human insulin, and the human insulin single-chain precursor has an amino acid sequence shown in SEQ ID NO. 1.
In another preferred embodiment, the weight ratio of human insulin to human insulin single-chain precursor is (0.5X 10)5-50x105): 1, preferably (1x 10)5-10x105)：1。
In an eighth aspect of the present invention, there is provided a method for preparing a hybridoma cell line producing the human insulin resistant single-chain precursor monoclonal antibody according to the first aspect of the present invention, comprising the following steps:
(1) using human insulin single-chain precursor as antigen, which is shown in SEQ ID NO.1, to immunize mammals;
(2) fusing the spleen cells of the immunized mammal with myeloma cells, and culturing; screening to obtain hybridoma cell strain secreting monoclonal antibody with specific reaction to the protein shown in SEQ ID No. 1.
In another preferred embodiment, the fusion ratio is splenocyte: the ratio of SP2/0 to SP 895/1 is 4.5-5.5.
The ninth aspect of the invention provides an enzyme-linked immunosorbent assay (ELISA) method for detecting a human insulin-containing single-chain precursor sample, which is characterized by comprising the following steps:
(1) coating the coated antibody on an ELISA plate, adding a human insulin single-chain precursor sample to form a coated antibody-human insulin single-chain precursor compound, wherein the human insulin single-chain precursor has an amino acid sequence shown in SEQ ID NO.1, and the coated antibody is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156;
(2) adding a detection antibody into the coated antibody-human insulin single-chain precursor compound to form a coated antibody-human insulin single-chain precursor-detection antibody compound, and detecting the human insulin single-chain precursor by detecting the coated antibody-human insulin single-chain precursor-detection antibody compound, wherein the detection antibody is a monoclonal antibody generated by a hybridoma cell line with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156, and the detection antibody and the coated antibody are different antibodies.
In another preferred embodiment, the method comprises a quantitative or qualitative method.
In another preferred embodiment, the method is an in vitro method.
In another preferred embodiment, the method is a non-diagnostic and/or non-therapeutic method.
In another preferred embodiment, the sample comprises a human insulin single chain precursor.
In another preferred embodiment, the sample contains a human insulin single-chain precursor and human insulin, and the human insulin single-chain precursor has an amino acid sequence shown in SEQ ID NO. 1.
In another preferred embodiment, the weight ratio of human insulin to human insulin single-chain precursor is (0.5X 10)5-50x105): 1, preferably (1x 10)5-10x105)：1。
In another preferred embodiment, the detection antibody is conjugated to a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or the like.
In another preferred embodiment, the enzyme comprises horseradish peroxidase.
In another preferred embodiment, the detection coating antibody-human insulin single chain precursor-detection antibody complex comprises the steps of:
adding an enzyme substrate into the coated antibody-human insulin single-chain precursor-detection antibody compound, and reading an OD value by an enzyme-labeling instrument;
wherein the detection antibody is coupled with an enzyme.
In another preferred embodiment, the method comprises the steps of:
coating the coated antibody on an ELISA plate, washing the plate, sealing, adding a human insulin single-chain precursor sample, incubating, adding a detection antibody to form a coated antibody-human insulin single-chain precursor-detection antibody complex, and detecting the human insulin single-chain precursor by detecting the coated antibody-human insulin single-chain precursor-detection antibody complex.
In another preferred embodiment, the method comprises the steps of:
coating the coated antibody on an ELISA plate, washing the plate, sealing, adding a human insulin single-chain precursor sample, incubating, adding a detection antibody coupled with horseradish peroxidase to form a coated antibody-human insulin single-chain precursor-detection antibody compound, washing the plate, adding an enzyme substrate (3,3',5,5' -tetramethylbenzidine), incubating, adding a stop solution, and reading the OD value of an ELISA reader.
The tenth aspect of the invention provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting a human insulin single-chain precursor sample, wherein the kit comprises:
the coating antibody is a monoclonal antibody produced by hybridoma cell lines with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156; and
the detection antibody is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156, and the detection antibody and the coating antibody are different antibodies.
In another preferred example, the enzyme-linked immunoassay kit further comprises a reaction plate (detection plate), the reaction plate is provided with micropores, and the micropores are coated or fixed with a coating antibody,
in another preferred example, the reaction plate is an enzyme label plate.
In another preferred embodiment, the enzyme-linked immunoassay kit further comprises a label or a specification, wherein the label or the specification indicates that the kit is used for detecting the human insulin single-chain precursor.
In another preferred embodiment, the sample comprises a human insulin single chain precursor.
In another preferred embodiment, the sample contains a human insulin single-chain precursor and human insulin, and the human insulin single-chain precursor has an amino acid sequence shown in SEQ ID NO. 1.
In another preferred embodiment, the weight ratio of human insulin to human insulin single-chain precursor is (0.5X 10)5-50x105): 1, preferably (1x 10)5-10x105)：1。
In an eleventh aspect of the present invention, there is provided an antibody combination, comprising:
the coating antibody is a monoclonal antibody produced by hybridoma cell lines with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156; and
the detection antibody is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156, and the detection antibody and the coating antibody are different antibodies.
In another preferred embodiment, the coating antibody and the detection antibody are independent,
in a twelfth aspect of the present invention, there is provided a use of the antibody combination of the eleventh aspect of the present invention for preparing a detection reagent or an enzyme-linked immunosorbent assay (ELISA) kit for detecting human insulin single-chain precursor.
In another preferred embodiment, the enzyme-linked immunoassay (ELISA) comprises a double-antibody sandwich ELISA.
In a thirteenth aspect of the present invention, there is provided an immune complex, comprising:
A-B-C
wherein A comprises a primary antibody, said primary antibody comprising the 17H3 antibody; the 17H3 antibody is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020138;
b comprises a human insulin single-chain precursor, and the human insulin single-chain precursor has an amino acid sequence shown in SEQ ID NO. 1;
the C comprises a secondary antibody, the secondary antibody comprises a 4B1 antibody, and the 4B1 antibody is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020156.
In another preferred embodiment, the primary antibody is conjugated to a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
In another preferred embodiment, the secondary antibody is conjugated to a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
In another preferred embodiment, the enzyme comprises horseradish peroxidase.
In another preferred embodiment, the human insulin single-chain precursor has an amino acid sequence shown in SEQ ID NO. 1.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Detailed Description
The inventor unexpectedly discovers an anti-human insulin single-chain precursor monoclonal antibody through long-term and intensive research and a large amount of screening, wherein the antibody is a monoclonal antibody generated by hybridoma cell lines with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156. The monoclonal antibody of the human insulin single-chain precursor is used as a coating antibody and a detection antibody for detecting the human insulin single-chain precursor in a sample by an ELISA method, and has the advantages of high sensitivity, good repeatability, good linearity, high accuracy and the like, so that the human insulin single-chain precursor in the sample can be effectively detected. On this basis, the inventors have completed the present invention.
Term(s) for
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the terms "comprising," "including," and "containing" are used interchangeably and include not only open-ended definitions, but also semi-closed and closed-ended definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".
As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the terms "human insulin single chain precursor" and "single chain precursor" are used interchangeably. Typically, the human insulin single-chain precursor comprises the amino acid sequence shown in SEQ ID NO: 1:
MVSKGEELFTGVKLTLKFICTTYVQERTISFKDTYKTRAEVKFEGDENLYFQGRFVNQHLCGSHLVEALYLVCGERGFFYTPK(Boc)TRGIVEQCCTSICSLYQLENYCN(SEQ ID NO:1)。
as used herein, the amino acid sequence of human insulin is SEQ ID NO 2.
FVNQHLCGSHLVEALYLVCGERGFFYTPKT-GIVEQCCTSICSLYQLENYCN(SEQ ID NO:2)。
As used herein, the amino acid sequence of the leader peptide is as shown in SEQ ID NO 3:
MVSKGEELFTGVKLTLKFICTTYVQERTISFKDTYKTRAEVKFEGDENLYFQGR(SEQ ID NO:3)
in the present invention, unless otherwise defined, amino acid sequences are numbered from N-terminus to C-terminus.
Anti-human insulin single chain precursor monoclonal antibody
The invention provides an anti-human insulin single-chain precursor monoclonal antibody which specifically recognizes and combines with a protein shown in SEQ ID NO. 1.
The term "specificity" as used herein refers to the ability of a monoclonal antibody to recognize a corresponding antigen (e.g., a protein) or a substance similar to the antigen. High specificity and strong recognition capability to antigen (such as protein). In the present invention, the monoclonal antibody against human insulin single-chain precursor can specifically recognize and bind to the protein shown in SEQ ID NO. 1. The protein shown in SEQ ID NO.1 can be directly synthesized or prepared by a genetic engineering method.
The human insulin single-chain precursor for preparing the monoclonal antibody of the present invention can be obtained by genetic engineering methods, and a polynucleotide containing a sequence encoding the human insulin single-chain precursor of the present invention is expressed in prokaryotes. Vectors suitable for expression of the human insulin single chain precursor of the present invention are generally well known to those skilled in the art. The appropriate vector can be selected according to the appropriate promoter selected and the gene sequence of interest to be expressed. Any suitable host cell may be used to express the human insulin single chain precursor of the present invention. Examples of suitable hosts include: prokaryotic cells such as E.coli, Bacillus, Streptomyces, and the like. Methods of transduction, transformation, or transfection are known in the art and include, but are not limited to, viral infection, calcium chloride transfection, lipofection, electroporation, or microparticle bombardment, among others. The transduced, transfected or transformed host cells can be cultured in conventional nutrient media, suitably modified, in order to activate promoters, select transformants or amplify the desired genes. The temperature, pH, etc. of the culture conditions used in the culture are generally determined by the host cell selected for expression of the particular protein, and are well known to those skilled in the art. In order to obtain a large amount of recombinant protein, an inducible promoter may be used, and the expression of the gene may be induced using an inducer. Essentially, the "inducer" can be any substance capable of inducing expression of a gene in a host, either a chemical or environmental stimulus.
Preferably, the anti-human insulin single-chain precursor monoclonal antibody is obtained by immunizing an animal by using a recombinant protein formed by a protein shown in SEQ ID NO.1 and a known label as an antigen. Preferably, the recombinant protein is used for immunizing a mouse, spleen cells of the mouse are taken and fused with myeloma cells, hybridoma cells capable of secreting monoclonal antibodies specifically reacting to the protein shown in SEQ ID NO.1 are screened, and the anti-human insulin single-chain precursor monoclonal antibodies are obtained from culture supernatants of the hybridoma cells or from ascites of animals injected with the hybridoma cells.
More preferably, the known tag is, but not limited to, a histidine tag (His tag), glutathione S-transferase (GST tag), Trx tag, S-tag, HA tag, HSV tag, Myc tag, or VSV-G tag. In a specific embodiment of the invention, the known tag is a GST tag, which forms a recombinant protein with the protein shown in SEQ ID NO. 1.
More preferably, the monoclonal antibody is obtained by immunizing a mouse with the recombinant protein as an antigen.
Preferably, the monoclonal antibody is produced by hybridoma cell strain with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156.
As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycan protein with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.
The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant regions. There are mainly 5 classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
As used herein, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the rabbit monoclonal antibodies herein are obtained by constructing full-length rabbit monoclonal antibody gene expression vectors by molecular biology methods after screening phage libraries, transferring the vectors into eukaryotic expression systems, and harvesting cell supernatants after culture, and are free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
The invention also comprises a monoclonal antibody with the corresponding amino acid sequence of the monoclonal antibody for resisting the protein shown in SEQ ID NO.1, a monoclonal antibody with the variable region chain of the monoclonal antibody for resisting the protein shown in SEQ ID NO.1, and other proteins or protein conjugates and fusion expression products with the chains. Specifically, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having light and heavy chains with hypervariable regions (complementarity determining regions, CDRs) so long as the hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous to the hypervariable regions of the light and heavy chains of the invention.
As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the monoclonal antibody or fragment thereof directed against the protein shown in SEQ ID No. 4. The invention also comprises a cell surface marker or antigen combined with the monoclonal antibody or the fragment thereof for resisting the protein shown in SEQ ID NO. 1.
The invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab')2 fragments, antibody heavy chains, antibody light chains.
The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having heavy and light chains with variable regions, provided that the variable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the variable regions of the heavy and light chains of the antibody of the invention.
In general, the antigen binding properties of an antibody can be described by 3 specific regions in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.
The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest, since at least some of them are involved in binding to an antigen. Thus, the invention includes those molecules having the light and heavy chain variable regions of a monoclonal antibody with CDRs that are more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDRs identified herein. The invention provides a hybridoma cell strain with a preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156 and a monoclonal antibody generated by the hybridoma cell strain, and a person skilled in the art can identify a CDR region, a complete amino acid sequence and a coding gene sequence of the monoclonal antibody by adopting the conventional technology in the field. Therefore, the antibody of the invention comprises the monoclonal antibody with the CDR regions which are the same as the monoclonal antibody produced by the hybridoma cell strain with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156.
The invention includes not only complete monoclonal antibodies, but also fragments of antibodies with immunological activity or recombinant proteins formed by antibodies and other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a recombinant protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.
The antibody of the present invention refers to a polypeptide having the binding activity to the protein represented by SEQ ID NO.1 and comprising the above-mentioned CDR regions. The term also includes variants of the polypeptides comprising the CDR regions described above that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.
Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
The invention also provides other polypeptides, such as recombinant proteins comprising human antibodies or fragments thereof. In addition to almost full-length polypeptides, the invention also encompasses fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.
In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.
TABLE A
The invention also provides a polynucleotide molecule for encoding the antibody or the fragment thereof or the recombinant protein thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.
The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.
The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more.
The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a recombinant protein.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.
At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.
Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, such as E.coli, competent cells capable of DNA uptake can be harvested after the exponential growth phase and treated by the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
Therapeutic agents that may be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 5. viral particles (Peng et al, 2004, Gene therapy 11, 1234); 6. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 7. nano magnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.
Coated antibodies
As used herein, "coated antibody," also known as a "capture antibody" or "solid phase antibody," refers to an antibody coated on a solid phase support. The coated antibody can be nonspecifically adsorbed or physically adsorbed on polystyrene (ELISA plate), nitrocellulose membrane, etc., to become solid phase antibody, and still maintain its immunological activity. The coating antibody has high affinity and high specificity to the antigen, but does not affect the binding of the antigen to the detection antibody. And adding a sample to be detected, and if the sample contains the target antigen, capturing the target antigen by the coated antibody. The antigen (antigen of interest) according to the present invention is preferably a human insulin single chain precursor. In a preferred embodiment of the invention, the coated antibody binds to the human insulin single-chain precursor with high affinity and high specificity. In another preferred example, the coating antibody is a monoclonal antibody produced by a hybridoma cell line with a preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156. Preferably, the coating antibody is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020138.
Detection of antibodies
As used herein, "detection antibody," also referred to as "labeled antibody," refers to an antibody used to detect whether a sample contains an antigen of interest. The detection antibody typically carries a detectable label, including a chromophore, a chemiluminescent group, a fluorophore, an isotope, or an enzyme. The detection antibody has high affinity and high specificity to the antigen, but does not affect the binding of the antigen to the coating antibody. The presence and quantity of the antigen is determined by detection of the label using the property of the detection antibody to bind specifically to the antigen of interest. The antigen (antigen of interest) according to the present invention is preferably a human insulin single chain precursor. In a preferred embodiment of the invention, the detection antibody binds to the human insulin single-chain precursor with high affinity and high specificity. In another preferred example, the detection antibody is a monoclonal antibody produced by a hybridoma cell line with a preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156. Preferably, the detection antibody is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020156.
Composition comprising a metal oxide and a metal oxide
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the antibody or active fragment thereof or recombinant protein thereof of the present invention, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
The pharmaceutical composition of the present invention can be directly used for binding human insulin single-chain precursor, and thus can be used for detecting human insulin single-chain precursor in a sample. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
Hybridoma cell strain
The invention provides two hybridoma cell lines which are characterized by being used for producing the anti-human insulin single-chain precursor monoclonal antibody, wherein the preservation number of the hybridoma cell line is CCTCC NO: C2020138 or CCTCC NO: C2020156, the antibody produced by the hybridoma cell line with the preservation number of CCTCC NO: C2020138 is 17H3 antibody (17H 3 for short), and the antibody produced by the hybridoma cell line with the preservation number of CCTCC NO: C2020156 is 4B1 antibody (4B 1 for short).
Labelled anti-human insulin single chain precursor monoclonal antibodies
In a preferred embodiment of the invention, the anti-human insulin single chain precursor monoclonal antibody is provided with a detectable label. More preferably, the detectable labels include (but are not limited to): a colloidal gold label, a colored label, or a fluorescent label.
The colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the present invention, the monoclonal antibody is labeled with colloidal gold to obtain a colloidal gold-labeled monoclonal antibody.
The monoclonal antibody of the invention has good specificity and high titer.
Enzyme-linked immunosorbent assay (ELISA) method
The monoclonal antibody of the human insulin single-chain precursor can be used as a coating antibody or a detection antibody in an enzyme-linked immunosorbent assay (ELISA) method and is used for qualitative or quantitative detection of the human insulin single-chain precursor.
Representatively, the invention relates to an enzyme-linked immunosorbent assay (ELISA) method for detecting a human insulin-containing single-chain precursor sample, which comprises the following steps:
(1) coating the coated antibody on an ELISA plate, adding a human insulin single-chain precursor sample to form a coated antibody-human insulin single-chain precursor compound, wherein the human insulin single-chain precursor has an amino acid sequence shown in SEQ ID NO.1, and the coated antibody is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156;
(2) adding a detection antibody into the coated antibody-human insulin single-chain precursor compound to form a coated antibody-human insulin single-chain precursor-detection antibody compound, and detecting the human insulin single-chain precursor by detecting the coated antibody-human insulin single-chain precursor-detection antibody compound, wherein the detection antibody is a monoclonal antibody generated by a hybridoma cell line with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156, and the detection antibody and the coated antibody are different antibodies.
In another preferred embodiment, the method comprises a quantitative or qualitative method.
In another preferred embodiment, the method is an in vitro method.
In another preferred embodiment, the method is a non-diagnostic and/or non-therapeutic method.
In another preferred embodiment, the detection antibody is conjugated to a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or the like.
In another preferred embodiment, the enzyme comprises horseradish peroxidase.
In another preferred embodiment, the detection coating antibody-human insulin single chain precursor-detection antibody complex comprises the steps of:
adding an enzyme substrate into the coated antibody-human insulin single-chain precursor-detection antibody compound, and reading an OD value by an enzyme-labeling instrument;
wherein the detection antibody is coupled with an enzyme.
In another preferred embodiment, the method comprises the steps of:
coating the coated antibody on an ELISA plate, washing the plate, sealing, adding a human insulin single-chain precursor sample, incubating, adding a detection antibody to form a coated antibody-human insulin single-chain precursor-detection antibody complex, and detecting the human insulin single-chain precursor by detecting the coated antibody-human insulin single-chain precursor-detection antibody complex.
In another preferred embodiment, the method comprises the steps of:
coating the coated antibody on an ELISA plate, washing the plate, sealing, adding a human insulin single-chain precursor sample, incubating, adding a detection antibody coupled with horseradish peroxidase to form a coated antibody-human insulin single-chain precursor-detection antibody compound, washing the plate, adding an enzyme substrate (3,3',5,5' -tetramethylbenzidine), incubating, adding a stop solution, and reading the OD value of an ELISA reader.
Reagent kit
The invention also provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting the human insulin single-chain precursor sample, which comprises the following components in percentage by weight:
the coating antibody is a monoclonal antibody produced by hybridoma cell lines with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156; and
the detection antibody is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156, and the detection antibody and the coating antibody are different antibodies.
In another preferred example, the enzyme-linked immunoassay kit further comprises a reaction plate (detection plate), the reaction plate is provided with micropores, and the micropores are coated or fixed with a coating antibody,
in another preferred example, the reaction plate is an enzyme label plate.
In another preferred embodiment, the enzyme-linked immunoassay kit further comprises a label or a specification, wherein the label or the specification indicates that the kit is used for detecting the human insulin single-chain precursor.
In another preferred embodiment, the sample comprises a human insulin single chain precursor.
In another preferred embodiment, the sample contains a human insulin single-chain precursor and human insulin, and the human insulin single-chain precursor has an amino acid sequence shown in SEQ ID NO. 1.
In another preferred embodiment, the weight ratio of human insulin to human insulin single-chain precursor is (0.5X 10)5-50x105): 1, preferably (1x 10)5-10x105)：1。
Immune complex
The present invention also provides an immune complex comprising:
A-B-C
wherein A comprises a primary antibody, said primary antibody comprising the 17H3 antibody; the 17H3 antibody is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020138;
b comprises a human insulin single-chain precursor, and the human insulin single-chain precursor has an amino acid sequence shown in SEQ ID NO. 1;
the C comprises a secondary antibody, the secondary antibody comprises a 4B1 antibody, and the 4B1 antibody is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020156.
In another preferred embodiment, the primary antibody is conjugated to a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
In another preferred embodiment, the secondary antibody is conjugated to a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
In another preferred embodiment, the enzyme comprises horseradish peroxidase.
In another preferred embodiment, the human insulin single-chain precursor has an amino acid sequence shown in SEQ ID NO. 1.
The main advantages of the invention include:
the human insulin single-chain precursor monoclonal antibody can be specifically combined with a human insulin single-chain precursor, and the human insulin single-chain precursor monoclonal antibody serving as a coating antibody and a detection antibody is used for detecting the human insulin single-chain precursor in a sample by an ELISA method, so that the human insulin single-chain precursor in the sample can be effectively detected.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Example 1
A human insulin single-chain precursor and a single-chain precursor can be used interchangeably, and the human insulin single-chain precursor has an amino acid sequence shown as SEQ ID NO: 1:
MVSKGEELFTGVKLTLKFICTTYVQERTISFKDTYKTRAEVKFEGDENLYFQGRFVNQHLCGSHLVEALYLVCGERGFFYTPK(Boc)TRGIVEQCCTSICSLYQLENYCN(SEQ ID NO:1)。
the DNA fragment of human insulin single-stranded precursor was cloned into NcoI-XhoI site downstream of araBAD promoter of expression vector plasmid pBAD/His A (purchased from NTCC, kanamycin resistance) to obtain plasmid pBAD-FP-TEV-R-MiniINS. The DNA sequence of pylRs was then cloned into the SpeI-SalI site downstream of the araBAD promoter of the expression vector plasmid pEvol-pBpF (available from NTCC for chloramphenicol resistance), while the DNA sequence of the tRNA (pylTcua) of lysyl-tRNA synthetase was PCR inserted downstream of the proK promoter. This plasmid was designated pEvol-pylRs-pylT.
The plasmid pBAD-FP-TEV-R-MiniINS and the plasmid pEvol-pylRs-pylT are transformed into an Escherichia coli strain, and the recombinant Escherichia coli strain expressing the Boc-human insulin single-chain precursor is obtained by screening. Then the single-chain precursor of human insulin is obtained through fermentation culture.
The amino acid sequence of human insulin is shown as SEQ ID NO: 2:
FVNQHLCGSHLVEALYLVCGERGFFYTPKT-GIVEQCCTSICSLYQLENYCN(SEQ ID NO:2)。
the amino acid sequence of the leader peptide is shown as SEQ ID NO. 3:
MVSKGEELFTGVKLTLKFICTTYVQERTISFKDTYKTRAEVKFEGDENLYFQGR(SEQ ID NO:3)。
1. principle of experiment
This example uses a double antibody sandwich ELISA: an enzyme-linked reaction plate is coated with a monoclonal antibody (a coating antibody and a primary antibody) of an anti-human insulin single-chain precursor, a sample to be detected containing the human insulin single-chain precursor is added for reaction, the non-binding substance is removed by washing, another monoclonal antibody (a detection antibody and a secondary antibody) of the anti-human insulin single-chain precursor marked by HRP (horse radish peroxidase) is added, a coating anti-human insulin single-chain precursor-HRP secondary marked antibody compound is formed after incubation, and the content of the single-chain precursor in the sample is indicated through the color development degree of TMB (3,3',5,5' -tetramethyl benzidine).
2. Preparation of anti-human insulin single chain precursor and evaluation of different antibody pairing screens
Respectively mixing a human insulin single-chain precursor, a leader peptide and an adjuvant to prepare two immunogens; 3 mice were immunized, respectively, and the tail blood was evaluated for antibody titer by an indirect ELISA (enzyme-linked immunosorbent assay) method.
Respectively coating an enzyme label plate (1 mu g/mL) with a human insulin single-chain precursor and a leader peptide, adding 100 mu L of the enzyme label plate into each hole, and reacting at 4 ℃ overnight; the plate was washed 3 times with PBS solution and blocked with 5% mil-PBS (phosphate buffered saline) for 1h at room temperature; then washing the plate for 1 time by using a PBS solution, adding the mouse tail blood diluted in a gradient manner, and reacting for 1 hour at room temperature; washing the plate for 3 times by using a PBS solution, beating to dry, adding a HRP-labeled goat anti-mouse Fc secondary antibody diluted by 1:2000, reacting for 1h at room temperature, washing the plate for 5 times by using the PBS solution, beating to dry, adding substrate A solution and substrate B solution with equal volume, and reacting for 20min at room temperature in a dark place; then adding 50 mu L of stop solution; the results of the indirect ELISA evaluation of mouse tail blood are shown in tables 1 and 2.
TABLE 1 evaluation of Single chain Procursor immunization group mouse tail blood antibody titers
Note: NC is negative control, PC is positive control 13# mouse tail blood.
TABLE 2 evaluation of tail blood antibody titers of leader peptide immunized mice
Note: NC is negative control, and PC is positive control 27# mouse tail blood.
The tail blood titer of the single-chain precursor immune group 30# mouse and the leader peptide immune group 27# mouse is highest, the immune mouse with the highest blood titer is selected, spleen cells are respectively taken to be fused with SP2/0 cells after being sacrificed, the spleen cells are cultured into monoclonal cell strains in a selective culture medium and then are selected to be cloned, and clones of 6 96-well plates are respectively selected. Respectively coating an enzyme label plate (1 mu g/mL) with a human insulin single-chain precursor and a leader peptide, adding 100 mu L of the enzyme label plate into each hole, and reacting at 4 ℃ overnight; the plate was washed 3 times with PBS solution and blocked with 5% mik-PBS for 1h at room temperature; the plate was then washed 1 time with PBS solution; in addition, the monoclonal cell supernatant and 5% mil-PBS 1:1 mixed, room temperature reaction for 1h, then add into the hole, then room temperature reaction for 1 h; washing the plate for 3 times by using PBS solution, beating to dry, adding HRP-labeled goat anti-mouse Fc secondary antibody diluted at a ratio of 1:2000, reacting for 1h at room temperature, washing the plate for 5 times by using PBS solution, beating to dry, adding the solution A and the solution B with equal volume, and reacting for 20min at room temperature in a dark place; then 50. mu.L of stop solution was added.
The single-chain precursor immunization group is used for primarily screening and reserving 9 positive clones with OD more than or equal to 1.0 from 564 clones in 6 96-well plates; the leader peptide immune group primarily screens 564 clones in 6 96-well plates to reserve 16 positive clones with OD more than or equal to 0.5; the results of the positive clone detection are shown in tables 3 and 4.
TABLE 3 Primary screening of Single-stranded precursors for Positive cloning test results
| Clone# | 4B1 | 3E4 | 5G1 | 5G6 | 6B8 | 4A2 | 4E6 | 4E11 | 6C4 | PC | NC |
| OD(450nm) | >4.000 | >4.000 | >4.000 | 3.132 | 2.515 | 2.481 | 2.003 | 1.138 | 1.018 | 3.119 | 0.021 |
Note: NC is negative control, and PC is positive control 30# mouse heart blood.
TABLE 4 leader peptide Primary screening Positive cloning test results
| Clone# | 12F3 | 11H3 | 9A8 | 12H7 | 12G10 | 12G6 | 12H5 | 12G7 | 12H4 | 9A3 | 8H8 | 12G5 |
| OD(450nm) | 3.688 | 2.127 | 1.868 | 1.843 | 1.350 | 1.328 | 0.992 | 0.970 | 0.817 | 0.654 | 0.563 | 0.526 |
| Clone# | 12E4 | 9A2 | 8F9 | 12H10 | PC | NC | ||||||
| OD(450nm) | 0.522 | 0.513 | 0.511 | 0.508 | >4.000 | 0.007 |
Note: NC is negative control, PC is positive control 27# mouse heart blood.
Rescreening the primary screened positive clones, wherein the detection results are shown in tables 5 and 6, and rescreening and retaining 3 positive clones for identifying the single-chain precursor from 9 primary screened positive clones of the fusion protein immune group; the leader peptide immunization group was screened for 16 positive clones and rescreened to retain 2 positive clones recognizing the leader peptide.
TABLE 5 Single-Strand precursor Primary screening Positive clone Re-screening test results
Note: NC is negative control, and PC is positive control 30# mouse heart blood.
TABLE 6 Positive cloning and rescreening test results of leader peptide prescreening
Note: NC is negative control, PC is positive control 27# mouse heart blood.
Performing specificity screening on the rescreened positive clones, wherein the detection results are shown in tables 7 and 8, 3E4 and 4B1 only recognize single-chain precursors and do not recognize leader peptides and insulin, and 5G1 recognizes single-chain precursors and insulin and does not recognize leader peptides in 3 positive clones which recognize single-chain precursors in a single-chain precursor immunization group; leader peptide immunization group 2 positive clones recognizing leader peptide all recognized single chain precursors and leader peptide, and did not recognize insulin.
TABLE 7 screening of Positive clone specificity for Single Strand precursor rescreening
Note: NC is negative control, and PC is positive control 30# mouse heart blood.
TABLE 8 Positive clone specificity screening by leader peptide rescreening
Note: NC is negative control, PC is positive control 27# mouse heart blood.
Selecting proper positive clone and its monoclonal antibody. Wherein the antibody generated by the hybridoma cell strain KPS 001-717H 3 with the preservation number of CCTCC NO: C2020138 is 17H3 antibody (17H 3 for short), and the antibody generated by the hybridoma cell strain KPS 001-64B 1 with the preservation number of CCTCC NO: C2020156 is 4B1 antibody (4B 1 for short).
The prepared different monoclonal antibodies are paired pairwise, the sensitivity for recognizing the human insulin single-chain precursor is detected by the double-antibody sandwich ELISA method, and the human insulin single-chain precursor is diluted into 1792, 896, 448, 224, 112, 56, 28, 14, 7 and 3.5ng/mL by using a sample diluent as a standard substance. As shown in Table 9, the two antibodies, i.e., the coated antibody 17H3, the detection antibody 4B1-HRP, the coated antibody 18F3 and the detection antibody 5G1-HRP, have obvious gradient on a standard curve, appropriate color development time and signal value, and are suitable for reagent kit development.
TABLE 9 different antibody pairing screens
3. Specificity of
After the standard curve concentration is adjusted (the concentration of the single-chain precursor of human insulin is adjusted to be 224, 112, 56, 28, 14, 7 and 3.5ng/mL), the sensitivity of two pairs of 17H3-4B1-HRP and 18F3-5G1-HRP is detected, and as shown in the following table 2, the sensitivity of the two pairs can reach 3.5 ng/mL. Human insulin was diluted to 5mg/mL, 5. mu.g/mL and 5ng/mL using sample dilutions and crossover of partner antibody with human insulin was detected. 18F3-5G1-HRP has cross with human insulin, 17H3-4B1-HRP has good specificity, only recognizes a human insulin single-chain precursor, does not recognize human insulin, and is shown in Table 10 in detail, so that the 17H3-4B1-HRP is used for matching.
TABLE 10 paired antibody sensitivity and specificity detection
Note: 17H3 and 18F3 are coating antibodies, and 4B1 and 5G1 are detection antibodies.
4. Performance determination of ELISA kit with 17H3 as coating antibody and 4B1-HRP as detection antibody
4.1 Using procedure for ELISA kits
Balancing: the desired reagents were transferred to room temperature (18-25 ℃) and equilibrated for 30 min.
Preparing liquid:
1 × diluent: taking 1 bottle of 20 Xdiluted solution, diluting to 1000ml with deionized water, and mixing well for later use.
1 × washing solution: 1 bottle of 20 XPBST (phosphate Tween buffer) washing solution is diluted to 1000ml by deionized water and is mixed for standby.
Sample dilutions (0.5% casein): casein (0.5 g/bag) was dissolved completely in 100ml of the prepared 1 Xdiluent solution and mixed well for use.
Enzyme conjugate dilutions (3% BSA): BSA (bovine serum albumin) (3 g/bag) was dissolved completely in the 1 Xdiluted solution and mixed well for use.
Enzyme conjugate working solution: the desired enzyme conjugate was diluted 100 times with a diluent prepared from an enzyme conjugate diluent (3% BSA), and mixed well for use.
Sample adding: taking out the coated plate from the sealed bag, adding 100ul of prepared standard substance and sample into each well, setting negative control, sealing the plate with sealing plate film, placing in a 37 deg.C constant temperature shaking incubator, and incubating at 200rpm for 60 min.
Washing: discarding liquid in each hole, filling micropores (350 mu L/hole) with 1 multiplied washing liquid, standing for 30 seconds, and discarding liquid in the hole; repeating for 6 times, and drying on the area paper after the last time of plate washing is finished.
Adding an enzyme conjugate working solution: add 100. mu.L of the working solution of the enzyme conjugate to each well, seal the wells with a sealing plate, put the wells in a 37 ℃ constant temperature shaking incubator at 200rpm, and incubate the wells for 60 minutes.
Washing: discarding liquid in each hole, filling micropores (350 mu L/hole) with 1 multiplied washing liquid, standing for 30 seconds, and discarding liquid in the hole; repeating for 6 times, and drying on the area paper after the last time of plate washing is finished.
Color development: adding 100 μ L of monocomponent color developing solution into each well, slightly shaking, mixing, sealing with sealing plate membrane, and developing at 25 deg.C for 10 min.
And (3) determination: add stop solution 50. mu.L to each well and mix gently. And selecting the dominant wavelength of the microplate reader to be 450nm, measuring the light absorption value (OD value) of each hole, and performing linear fitting calculation by adopting a double-logarithm fitting mode.
4.2 validation of accuracy and precision
And (3) producing a coated plate by using the monoclonal antibody 17H3, detecting the antibody to be 4B1-HRP, determining the concentration of a quality control product according to a quantitative range, and verifying the accuracy and precision. The standard curve is shown in Table 11, and the accuracy and precision analysis results of the quality control products are shown in Table 12.
TABLE 11 Standard Curve
TABLE 12 accuracy and precision of quality control
As can be seen from tables 11 and 12, the linear range of the kit (the coating antibody is 17H3, and the detection antibody is 4B1-HRP) is 5-160 ng/mL, the accuracy RE% of each concentration quality control product (the single-chain precursor with different concentrations is diluted in 5mg/mL human insulin (human insulin is diluted by using a sample diluent)) is-19.97% -2.08%, the precision CV% is 0.34% -11.56%, and the acceptance standard is met: the accuracy (RE%) +/-20% and the precision (CV%) < 20%.
3.5 detection of test article (human insulin)
In the ELISA method, 17H3 is a coating antibody coated on an ELISA plate, and 4B1 is a detection antibody.
The date numbers are 1, 2 and 3 respectively, the numbers of analysts are Y and Q respectively, and the numbers of kits are A and B (different numbers of kits in the same batch). The experimental arrangement is shown in Table 13.
Single-chain precursor standard solution: 0ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 120ng/mL, respectively.
Each analyst tests 1 time a day, and adds a certain amount of standard solution to the sample solution so that the concentrations of the standard in the sample solution are 10ng/mL (low, 20% standard), 50ng/mL (medium, 100% standard) and 100ng/mL (high, 200% standard), respectively (3 parts for each standard concentration sample).
Test solution: precisely weighing a sample 50mg to 10mL in a volumetric flask, adding 1 Xdiluent to dissolve the sample, diluting to a scale, and shaking up to obtain the test solution (two prepared parts).
Adding a standard test sample stock solution: a sample (60 mg) is precisely weighed into a 10mL volumetric flask, and 1 Xdilution is added to dissolve and dilute the sample to the scale.
Preparing a standard-added low-concentration standard test solution: 417. mu.L of the stock solution of the test sample and a centrifuge tube were precisely pipetted, 10. mu.L of 500ng/mL standard solution and 73. mu.L of 1 Xdilution were added, and mixed well (preparation of 3 parts).
Preparing a test solution with a medium concentration standard in the added standard: 417. mu.L of the stock solution of the test sample and a centrifuge tube were precisely pipetted, 50. mu.L of 500ng/mL standard solution and 33. mu.L of 1 Xdilution were added, and mixed (3 parts).
Adding a standard test sample solution with high concentration for preparation: 834. mu.L of the stock solution of the test sample and the centrifuge tube are precisely transferred, 100. mu.L of 1000ng/mL standard solution and 66. mu.L of 1 Xdilution are added, and mixed (3 parts are prepared).
Calculating the RSD of the recovery rate of each specification concentration point, the RSD of the recovery rates of three specification concentration points and the RSD of the recovery rate of three days, wherein the RSD is less than or equal to 25.0 percent.
The recovery was calculated as follows:
in the formula:
c1- -is the measured concentration (ng/mL) of the added standard substance calculated by the regression equation;
c2- -is the theoretical concentration (ng/mL) at which the standard was added.
Cp- - -calculating the concentration (ng/mL) of the single-chain precursor of the test solution obtained by the regression equation;
the results of daily measurement are shown in tables 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25, and the results of intermediate precision are shown in Table 26.
TABLE 13 intermediate precision experimental arrangement
TABLE 14 day 1 Standard test results
TABLE 15 test results for day 1 test article
TABLE 16 test results of day 1 spiked test samples
TABLE 17 test results of recovery from day 1 spiking
Table 18 day 2 standard test results
TABLE 19 test results for day 2 test article
TABLE 20 test results of the 2 nd day spiked test samples
TABLE 21 test results of recovery from day 2 spiking
TABLE 22 day 3 Standard test results
TABLE 23 test results for test article on day 3
TABLE 24 test results of the 3 rd day spiked test samples
TABLE 25 test results of recovery from day 3 spiking
TABLE 26 intermediate precision results
The recovery rates of RSD of different specification concentration points on the 1 st day are respectively 7.1%, 4.5% and 8.5%, and the RSD of the three specifications is 12.2% and is less than 25.0%; the recovery rates of RSD of different specification concentration points at day 2 are respectively 5.1%, 3.7% and 2.4%, and the RSD of the three specifications is 22.7%, and both are less than 25.0%; the recovery rates of RSD of different specification concentration points on the 3 rd day are respectively 9.7%, 8.7% and 4.5%, and the RSD of the three specifications is 21.1% and is less than 25.0%; the recovery rates of RSD of different specification concentration points are respectively 6.6%, 5.7% and 6.3% in 3 consecutive days, and the total RSD of the three specifications is 1.5%, and is less than 25.0%; therefore, the intermediate precision is within the specification.
Strain preservation
The hybridoma cell strain KPS 001-717H 3 for generating the monoclonal antibody of the human insulin resistant single-chain precursor has been preserved in China center for type culture Collection (CCTCC, China, Wuhan), and the preservation date is as follows: 16/09/2020, accession No.: CCTCC NO: C2020138.
The hybridoma cell strain KPS 001-64B 1 for generating the monoclonal antibody of the human insulin resistant single-chain precursor has been preserved in China center for type culture Collection (CCTCC, China, Wuhan), and the preservation date is as follows: 16/09/2020, accession No.: CCTCC NO: C2020156.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Suzhou spread biotechnology, Inc
<120> detection control method for human insulin single-chain precursor residue
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Claims (10)
1. An anti-human insulin single-chain precursor monoclonal antibody is characterized in that the anti-human insulin single-chain precursor monoclonal antibody specifically recognizes and combines with a protein shown as SEQ ID NO. 1.
2. The monoclonal antibody of human insulin single chain precursor of claim 1, wherein the human insulin single chain precursor is a monoclonal antibody produced by hybridoma cell line with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156.
3. A hybridoma cell strain, which is used for producing the anti-human insulin single-chain precursor monoclonal antibody of claim 1, and has a preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156.
4. A recombinant protein, said recombinant protein having:
(i) an anti-human insulin single chain precursor monoclonal antibody according to claim 1; and
(ii) optionally a tag sequence to facilitate expression and/or purification.
5. An immunoconjugate, comprising:
(a) an anti-human insulin single chain precursor monoclonal antibody according to claim 1; and
(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
6. A pharmaceutical composition comprising:
(i) an anti-human insulin single chain precursor monoclonal antibody according to claim 1, or a recombinant protein according to claim 4, or an immunoconjugate according to claim 5; and
(ii) a pharmaceutically acceptable carrier.
7. Use of the monoclonal antibody of claim 1, the recombinant protein of claim 4, or the immunoconjugate of claim 5 for the preparation of a medicament, a reagent, a test plate, or a kit;
the reagent, assay plate or kit is for:
(1) the protein shown as SEQ ID NO.1 in the detected sample.
8. A method for preparing hybridoma cell line producing the anti-human insulin single-chain precursor monoclonal antibody of claim 1, comprising the following steps:
(1) using human insulin single-chain precursor as antigen, which is shown in SEQ ID NO.1, to immunize mammals;
(2) fusing the spleen cells of the immunized mammal with myeloma cells, and culturing; screening to obtain hybridoma cell strain secreting monoclonal antibody with specific reaction to the protein shown in SEQ ID No. 1.
9. An enzyme-linked immunosorbent assay (ELISA) method for detecting a human insulin-containing single-chain precursor sample, which is characterized by comprising the following steps:
(1) coating the coated antibody on an ELISA plate, adding a human insulin single-chain precursor sample to form a coated antibody-human insulin single-chain precursor compound, wherein the human insulin single-chain precursor has an amino acid sequence shown in SEQ ID NO.1, and the coated antibody is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156;
(2) adding a detection antibody into the coated antibody-human insulin single-chain precursor compound to form a coated antibody-human insulin single-chain precursor-detection antibody compound, and detecting the human insulin single-chain precursor by detecting the coated antibody-human insulin single-chain precursor-detection antibody compound, wherein the detection antibody is a monoclonal antibody generated by a hybridoma cell line with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156, and the detection antibody and the coated antibody are different antibodies.
10. An enzyme-linked immunosorbent assay (ELISA) kit for detecting a human insulin single-chain precursor sample, which is characterized by comprising:
the coating antibody is a monoclonal antibody produced by hybridoma cell lines with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156; and
the detection antibody is a monoclonal antibody produced by a hybridoma cell line with the preservation number of CCTCC NO: C2020138 or CCTCC NO: C2020156, and the detection antibody and the coating antibody are different antibodies.
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| CN117169518A (en) * | 2023-11-03 | 2023-12-05 | 赛德特(北京)生物工程有限公司 | Method and kit for detecting CD3 antibody residues in T lymphocyte preparation |
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| US5514599A (en) * | 1989-09-23 | 1996-05-07 | Hoechst Aktiengesellschaft | Antibodies against highly conserved amino acid sequences of insulin a process for the preparation of these antibodies and the use thereof in immunoassays |
| US20050037448A1 (en) * | 1997-06-20 | 2005-02-17 | Pasteur Sanofi Diagnostics | C-peptide specific assay procedure |
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| CN117169518A (en) * | 2023-11-03 | 2023-12-05 | 赛德特(北京)生物工程有限公司 | Method and kit for detecting CD3 antibody residues in T lymphocyte preparation |
| CN117169518B (en) * | 2023-11-03 | 2024-01-19 | 赛德特(北京)生物工程有限公司 | Method and kit for detecting CD3 antibody residues in T lymphocyte preparation |
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