CN116143929A - Antibody against recombinant human coagulation factor VIIa-Fc fusion protein and application thereof - Google Patents

Abstract

The present application provides an antibody or antigen binding fragment thereof that binds to a recombinant human factor VIIa-Fc fusion protein, and uses thereof for detecting the presence or amount of a recombinant human factor VIIa-Fc fusion protein in a sample.

Description

Antibody against recombinant human coagulation factor VIIa-Fc fusion protein and application thereof

Technical Field

The application relates to the technical field of biological medicine, in particular to a monoclonal antibody of an anti-recombinant human blood coagulation factor VIIa-Fc fusion protein and application thereof.

Background

Accurate determination of drug concentration in biological matrices (e.g., whole blood, serum, plasma, urine) is important for drug and formulation development, and these data can be used to support the safety and efficacy of drugs or to make critical decisions based on the results of efficacy tests of toxicology, pharmacokinetics, and biology, among others. Thus, the applied bioanalytical methods must be completely validated and documented to obtain reliable results.

The immunoassay method is an analytical method based on a specific antigen-antibody reaction. The immunoassay technology is combined with the radionuclide tracing technology, the enzymatic reaction or the fluorescent analysis and other high-sensitivity analysis technologies, has the characteristics of high specificity and high sensitivity, and is particularly suitable for measuring trace components in a complex system and rapidly measuring active ingredients in fermentation liquor or cell culture liquor in drug production. The key of the immunoassay method is to screen the antibody which is high-efficiency, sensitive and specific to the drug to be tested.

Patent ZL201610692686.4 discloses recombinant human coagulation factor vila-Fc fusion proteins, which connect FVIIa and half-life extending part human IgG Fc by using a linker molecule composed of a flexible peptide and CPT, and can retain the biological activity of FVIIa to the greatest extent and extend the in vivo activity half-life of FVIIa more remarkably.

Biological assays for preclinical and clinical studies have not been reported for recombinant human factor VIIa-Fc fusion proteins.

Disclosure of Invention

The purpose of the application is to provide a monoclonal antibody capable of being used for detecting recombinant human coagulation factor VIIa-Fc fusion protein and application thereof.

The application provides the following technical scheme for solving the technical problems:

in a first aspect the present application provides an antibody or antigen binding fragment thereof that binds a recombinant human factor VIIa-Fc fusion protein comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3; wherein, the liquid crystal display device comprises a liquid crystal display device,

the HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6; or alternatively

The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12.

In a second aspect the present application provides a polynucleotide encoding an antibody or antigen binding fragment thereof provided in the first aspect of the present application; or, a recombinant vector comprising said polynucleotide; or, a host cell comprising said polynucleotide or recombinant vector; or, a hybridoma cell for expressing an antibody or antigen-binding fragment thereof provided in the first aspect of the present application.

In a third aspect, the present application provides the use of an antibody or antigen-binding fragment thereof of the first aspect of the present application for detecting the presence or amount of a recombinant human factor VIIa-Fc fusion protein in a sample.

In a fourth aspect, the present application provides a method for detecting the presence or amount of recombinant human factor VIIa-Fc fusion protein in a sample using an antibody or antigen-binding fragment thereof of the first aspect of the present application, wherein the method is a sandwich ELISA method, the antibody or antigen-binding fragment thereof being used as a capture antibody and/or a detection antibody.

In a fifth aspect the present application provides an antibody combination for use in the detection of a recombinant human factor VIIa-Fc fusion protein comprising two of the antibodies or antigen binding fragments thereof provided in the first aspect of the present application.

In a sixth aspect the present application provides a kit for detecting a recombinant human factor VIIa-Fc fusion protein comprising at least one of the antibodies or antigen binding fragments thereof provided in the first aspect of the present application.

The beneficial effects are that:

the present application provides an antibody or antigen binding fragment thereof against recombinant human factor VIIa-Fc fusion protein, which can be used to detect the presence or amount of recombinant human factor VIIa-Fc fusion protein in a sample by specifically binding to recombinant human factor VIIa-Fc fusion protein. Further, the antibody or the antigen binding fragment thereof can specifically bind to different epitopes of the factor VIIa-Fc fusion protein, so that the antibody can be used as a capture antibody and/or a detection antibody for detecting the factor VIIa-Fc fusion protein, and the detection method of the factor VIIa-Fc fusion protein established by adopting the antibody has high stability, accuracy, precision, sensitivity, specificity and the like.

Detailed Description

The following description of the technical solutions in the embodiments of the present application will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.

Abbreviation table

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Definition of the definition

Unless otherwise indicated, practice of the present application will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

In order that the present application may be more readily understood, certain technical and scientific terms are specifically defined as follows. Unless otherwise defined explicitly herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. For definitions and terms in the art, the expert may refer specifically to Current Protocolsin Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. As used herein (including the claims), the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ± 5%, ±2%, ±1% and ± 0.1%.

The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the reference amino acid sequence after aligning the amino acid sequences (and introducing gaps, if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment may be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to obtain the maximum alignment for the full length of sequences compared.

The term "antibody" refers to any form of antibody that has the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies, and the like. The basic antibody structural unit is known to comprise tetramers, each comprising two identical pairs of polypeptide chains, each pair having one "light" chain (L, about 25 kDa) and one "heavy" chain (H, about 50-70 kDa). The amino-terminal portion or fragment of each chain may include a variable region of about 100-110 amino acids or more that is primarily responsible for antigen recognition. The carboxy-terminal portion or fragment of each strand may define a constant region primarily responsible for effector function. Human light chains are generally classified as kappa light chains and lambda light chains. Furthermore, human heavy chains are generally classified into five classes μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively, depending on the heavy chain. Within the light and heavy chains, the respective variable and constant regions are linked by a "J" region of about 12 or more amino acids, wherein the heavy chain further comprises a "D" region of about 10 more amino acids. See generally chapter 7 of Fundamental Immunology (Paul, W. Main, 2 nd edition, raven Press, N.Y. (1989)).

The term "isolated antibody" with respect to an antibody means that the antibody is substantially free of other cellular components associated with its natural state, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth media. It is understood that the isolated antibody is in a substantially purified state, preferably in a homogeneous state, and may be in a dry or aqueous solution. Analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography can be used to determine the purity and homogeneity of antibodies. The term "isolated" does not mean that the above-mentioned substances are completely absent or that water, buffer or salt are absent unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the antibodies of the present application.

The term "monoclonal antibody" is an antibody made from highly identical immune cells, which are all clones of a single parent cell. Monoclonal antibodies have monovalent affinity because they bind to the same epitope (the site where the antibody recognizes the antigen). The monoclonal antibodies may also include minor amounts of naturally occurring mutations. In contrast, the term "polyclonal antibody" binds to multiple epitopes, typically consisting of several different plasma cell (antibody secreting immune cell) lineages, and is understood to be a hybrid of multiple monoclonal antibodies. The modifier "monoclonal" is not to be construed as requiring antibody production by any particular method.

The term "binding domain" or "antigen binding site" refers to a region in an antibody that is capable of specifically binding to and complementing a portion or all of an antigen. When the antigen is large, the antibody may only bind to a specific portion of the antigen, which portion is referred to as an epitope. The binding domain may comprise the variable domains of the heavy and light chains, namely the heavy chain variable region VH and the light chain variable region VL, each comprising four conserved Framework Regions (FR) and three Complementarity Determining Regions (CDRs). CDRs can vary in sequence and determine specificity for a particular antigen.

The term "full length" antibody refers to an immunoglobulin molecule that when naturally occurring comprises four peptide chains: two heavy chains (full length about 50-70 kDa) and two light chains (full length about 25 kDa) are linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of 3 domains, CH1, CH2 and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region consists of one domain CL. VH and VL regions can be further subdivided into Complementarity Determining Regions (CDRs) with high variability and Framework Regions (FR) with higher conservation that are distributed with complementarity determining regions. The domains of each VH or VL from amino terminus to carboxy terminus are arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable domains of the heavy and light chains each contain a binding domain that interacts with an antigen. The constant region of an antibody may mediate the binding of the antibody to various cells of the host's tissue or immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

The term "heavy chain constant region" or "CH" is used interchangeably herein and comprises at least three heavy chain constant domains (CH 1, CH2 and CH 3). Illustratively, the human heavy chain constant regions include gamma, delta, alpha, epsilon, and mu, each heavy chain constant region corresponding to an antibody isotype. For example, the antibody comprising a gamma constant region is an IgG antibody, the antibody comprising a delta constant region is an IgD antibody, the antibody comprising an alpha constant region is an IgA antibody, the antibody comprising a mu constant region is an IgM antibody, and the antibody comprising an epsilon constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses, for example, igG antibodies include, but are not limited to, igG1 (comprising a γ1 constant region), igG2 (comprising a γ2 constant region), igG3 (comprising a γ3 constant region), and IgG4 (comprising a γ4 constant region); igA antibodies include, but are not limited to IgA1 (comprising an α1 constant region) and IgA2 (comprising an α2 constant region); igM antibodies include, but are not limited to, igM1 and IgM2. Isoforms may also include some form of modification that may alter Fc function, e.g., enhance or attenuate effector function or enhance or attenuate binding to Fc receptors.

The term "light chain constant region" or "CL" is used interchangeably herein and comprises 1 light chain constant domain CL. Illustratively, light chains can be classified into two classes, λ and κ, depending on the constant region of the light chain.

The term "antigen-binding fragment" of an antibody includes fragments of the antibody or derivatives of the antibody, and the antibody corresponding to the "antigen-binding fragment" may be referred to as a parent antibody. The antigen-binding fragment of an antibody typically comprises at least one fragment of the antigen-binding or variable region of the parent antibody that retains at least some of the binding specificity of the parent antibody. Examples of antigen binding fragments include, but are not limited to, fab ', F (ab') 2, and single chain Fv fragments, diabodies, linear antibodies, domain antibodies, single chain antibody molecules, e.g., scFv; nanobodies (nanobodies) and multispecific antibodies formed from antibody fragments, and the like. The antigen binding fragment is capable of retaining at least 10%, at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding activity of the parent antibody at the same molar concentration. Furthermore, an antigen-binding fragment of an antibody may also include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "functional conservative variants" of the antibody).

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term "nucleic acid" or "polynucleotide" also includes nucleic acids comprising known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Pat. No.8278036 to Kariko et al, which discloses mRNA molecules with uridine replaced by pseudouridine, methods of synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single Nucleotide Polymorphisms (SNPs) and complementary sequences, as well as the sequence explicitly indicated.

"construct" refers to any recombinant polynucleotide molecule (e.g., plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, linear or circular single-or double-stranded DNA or RNA polynucleotide molecule) that can be derived from any source, capable of integration with the genome or autonomous replication, which can be operably linked to one or more polynucleotide molecules. In this application, constructs are generally polynucleotide molecules of the present application operably linked to transcriptional initiation regulatory sequences that direct transcription of the polynucleotide molecules of the present application in a host cell. Heterologous promoters or endogenous promoters may be used to direct expression of the nucleic acids of the present application.

"vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introduction of heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, which can ligate additional DNA segments into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and are replicated along with the host genome.

The term "expression vector" as used herein refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell. Expression vectors typically contain one or more phenotypic selectable markers and an origin of replication for maintenance of the vector and amplification in the host if desired.

In a first aspect the present application provides an antibody or antigen binding fragment thereof that binds to a recombinant human factor VIIa-Fc fusion protein comprising three heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3 and three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3; wherein, the liquid crystal display device comprises a liquid crystal display device,

the HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6; or alternatively

The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12.

In this application, the recombinant human factor VIIa-Fc fusion protein (rhFVIIa-Fc) comprises FVIIa, a linker molecule (linker) and a human IgG Fc portion, having the amino acid sequence shown in SEQ ID NO. 21. Wherein the FVIIa amino acid sequence is shown as SEQ ID NO. 22, and the linker-Fc part amino acid sequence is shown as SEQ ID NO. 23.

For the precise amino acid sequence boundaries of the CDRs of an antibody, they can be defined according to well-known methods, e.g., chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al, nature 342:877-883, 1989; al-Lazikani et al, journal of Molecular Biology,273:927-948, 1997); or Kabat based on antibody sequence variability (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition, U.S. Pat. No. of Health and Human Services, national Institutes of Health, 1987), abM (University of Bath), contact (University College London), and IMGT (the international ImMunoGeneTics database,1999Nucleic Acids Research,27,209-212); or on the North CDR definition of a neighbor-propagated cluster (affinity propagation clustering) that utilizes a large number of crystal structures. The CDRs of the antibodies herein can be bordered by one of skill in the art according to any protocol in the art (e.g., the optional definition methods described above).

It should be noted that the boundaries of CDRs of the same antibody obtained based on different definitions may differ, i.e. the CDR sequences of the variable regions of the same antibody obtained under different definitions may differ. Thus, when an antibody is defined using a particular CDR sequence as defined herein, the antibody also includes antibodies whose complementarity determining region sequences comprise the CDRs as described herein, except that the stated CDR boundaries differ from the particular CDR boundaries as defined herein due to the use of different CDR boundary definitions.

Antibodies with different specificities (i.e., for different antigen combining sites) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding, and the minimal overlap region of an antibody CDR with antigen binding is also referred to as the "minimal binding unit" for antigen binding, and can be determined using at least two of Kabat, chothia, abM, contact and North methods. The minimum binding unit may be part of a CDR. As will be apparent to those skilled in the art, by the structure and protein folding of the antibody, the residues of the remainder of the CDR sequence may be determined, and thus, the present application also contemplates variants of any CDR, e.g., in a variant of one CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues according to the Kabat or Chothia definition may be replaced by conserved amino acid residues.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 13 and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 14; or (b)

A heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 15, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 16.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 13, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 14; or (b)

A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 15, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 16.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 17 and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 18; or (b)

A heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 19, and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 20.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

a heavy chain having an amino acid sequence shown as SEQ ID NO. 17, and a light chain having an amino acid sequence shown as SEQ ID NO. 18 (antibody 13G7B 9); or (b)

A heavy chain having an amino acid sequence shown in SEQ ID NO. 19, and a light chain (antibody 12E1B 6) having an amino acid sequence shown in SEQ ID NO. 20.

In a second aspect, the present application provides a polynucleotide encoding an antibody or antigen binding fragment thereof provided in the first aspect of the present application.

In some embodiments, the polynucleotide comprises at least one of a nucleotide sequence encoding an antibody or antigen binding fragment thereof provided herein or a complement thereof. Polynucleotides in this application include double-stranded or single-stranded DNA or RNA.

In some embodiments, a polynucleotide molecule encoding an antibody or antigen-binding fragment thereof of the present application may also include a polynucleotide sequence that has nucleotide deletions, insertions, or substitution mutations, but which still has at least about 60%, 70%, 80%, 90%, 95%, or 100% identity to the coding region corresponding to the CDRs of the antibody or antigen-binding fragment thereof of the present application.

In yet another aspect, the present application provides a recombinant vector comprising the polynucleotide. Preferably, the recombinant vector is a eukaryotic expression vector.

In yet another aspect, the present application provides a host cell comprising the polynucleotide or recombinant vector; preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

In some embodiments, the host cell is used to express an antibody or antigen-binding fragment thereof provided herein.

Mammalian host cells provided herein for expressing the antibodies or antigen binding fragments thereof of the present application include a variety of immortalized cell lines available from the American Type Culture Collection (ATCC). Exemplary, may include Chinese Hamster Ovary (CHO) cells, NS0, SP2/0 cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells, a549 cells, 293T cells, and many other cell lines. Mammalian host cells may include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. One skilled in the art can select particularly preferred cell lines by determining which cell lines have high expression levels.

In yet another aspect, the present application also relates to a hybridoma cell for expressing the antibody or antigen binding fragment thereof provided in the first aspect of the present application, which can be formed by fusion of B lymphocytes and myeloma cells of an antigen-immunized mouse.

In a third aspect, the present application provides the use of an antibody or antigen-binding fragment thereof of the first aspect of the present application for detecting the presence or amount of a recombinant human factor VIIa-Fc fusion protein in a sample.

In a fourth aspect, the present application provides a method of detecting the presence or amount of a recombinant human factor VIIa-Fc fusion protein in a sample using an antibody or antigen-binding fragment thereof of the present application, comprising contacting the antibody or antigen-binding fragment thereof with the sample, detecting a conjugate formed by the antibody or antigen-binding fragment thereof and the recombinant human factor VIIa-Fc fusion protein, and optionally quantifying the conjugate. The term "detection" as used in this application may include quantitative or qualitative detection. In some embodiments, the sample is a biological sample. In certain embodiments, the biological sample is whole blood, serum, plasma, urine, or other liquid sample of biological origin. The method of "quantifying the conjugate" is not limited in this application.

In some embodiments, the method of detecting the presence or amount of recombinant human factor VIIa-Fc fusion protein in a sample is an immunoassay, such as a radioimmunoassay, an enzyme-linked immunosorbent assay, an electrochemiluminescent immunoassay, or an immunofluorescent assay, among others.

In some embodiments, the method may be a sandwich ELISA method.

In some embodiments, the antibody or antigen binding fragment thereof serves as a capture antibody and/or a detection antibody.

For example, in some embodiments, an antibody or antigen-binding fragment thereof of the present application may be used as a capture antibody for capturing recombinant human factor VIIa-Fc fusion protein in a sample. In other embodiments, another antibody or antigen-binding fragment thereof of the present application may also be employed as a detection antibody for detecting recombinant human factor VIIa-Fc fusion protein captured by the capture antibody.

In some embodiments, the detection antibody may be a labeled antibody or antigen-binding fragment, and the "label" may be understood as the antibody or antigen-binding fragment is linked to a label; the label is a label commonly used in the art for detection, such as horseradish peroxidase (HRP), biotin (Biotin), or a fluorescent tag (e.g., GFP, FAM, VIC, CY3, etc.). Those skilled in the art will be able to match the desired label according to the detection method, and the present application is not limited thereto. Furthermore, the attachment of the label to the antibody is a conventional means in the art, and the present application is not limited thereto.

In some embodiments, the recombinant human factor VIIa-Fc fusion protein in the sample is captured using antibody 13G7B9 as a capture antibody; the labeled antibody 12E1B6 is used as a detection antibody for detecting the existence or the content of the recombinant human blood coagulation factor VIIa-Fc fusion protein captured by the capture antibody.

In a fifth aspect the present application provides an antibody combination for use in the detection of a recombinant human factor VIIa-Fc fusion protein comprising two of the antibodies or antigen binding fragments thereof provided in the first aspect of the present application.

In some embodiments, the antibody combination comprises antibody 13G7B9 and antibody 12E1B6.

In a sixth aspect the present application provides a kit for detecting a recombinant human factor VIIa-Fc fusion protein comprising at least one of the antibodies or antigen binding fragments thereof provided in the first aspect of the present application.

In some embodiments, the kit comprises a combination of antibodies of the present application; preferably, antibody 13G7B9 and antibody 12E1B6 are comprised.

Antibodies of the present application and their use are illustrated below by specific examples. It will be apparent that the described embodiments are only some, but not all, of the embodiments of the present application. All other embodiments obtained by those skilled in the art based on the embodiments herein fall within the scope of the protection of the present application.

EXAMPLE 1 preparation of monoclonal antibodies

1. Antigen preparation

The antigen used for immunization of animals in this example is rhFVIIa-Fc fusion protein, the amino acid sequence of which is shown in SEQ ID NO. 21, produced by Chengsi, zhengzhou, and the preparation method of which can be seen in patent ZL201610692686.4.

Antigens used in the detection of the antibodies of this example include: rhFVIIa-Fc fusion protein (with the sequence shown as SEQ ID NO: 21), FVIIa (with the amino acid sequence shown as SEQ ID NO: 22), linker-Fc (with the amino acid sequence shown as SEQ ID NO: 23). The antigens described above can be prepared by conventional methods known in the art, and can also be prepared and purified by methods disclosed in, for example, patent ZL201610692686.4.

2. Immunization of animals

5 BALB/c mice were immunized with the antigen rhFVIIa-Fc fusion protein of step 1 according to the immunization protocol of Table 1.

TABLE 1 immunization protocol

Flow name	Time	Immunization dose
			First immunization	T=0 days	50 μg/each
Second immunization	T=14 days	25 μg/each
			Third immunization	T=28 days	25 μg/each
Last immunization	T=50±7 days	25 μg/each

Blood was collected at t=21 days and t=35 days, and antibody titers in serum were detected by ELISA method with rhFVIIa-Fc fusion protein, linker-Fc, FVIIa, respectively, wherein the coating concentration of antigen was 1 μg/ml,100 μl/well; the coating buffer was PBS (pH 7.4); the secondary antibody was HRP-labeled goat anti-mouse IgG.

3. Cell fusion

(1) Selecting 1 mouse with the best antibody titer, taking out spleen, preparing spleen lymphocytes, and respectively fusing with SP2/0 myeloma cells to obtain hybridoma cells.

(2) And (3) primary screening: the supernatant of the fused cells was screened by indirect ELISA and the supernatant positive (+) for the rhFVIIa-Fc fusion protein was selected.

(3) Confirmatory screening: screening 5 strains of hybridoma cells specific for FVIIa from the hybridoma cell supernatant obtained by the primary screening by ELISA method [ i.e. rhFVIIa-Fc fusion protein positive (+) and FVIIa positive (+) and linker-Fc negative (-) ]; and 5 lines of hybridoma cells specific for linker-Fc [ i.e., rhFVIIa-Fc fusion protein positive (+) and linker-Fc positive (+) and FVIIa negative (-) ]. ELISA detection results of hybridoma cell supernatants are shown in Table 2, wherein the coating antigen is: a, rhFVIIa-Fc fusion protein; b, linker-Fc; c, FVIIa; the amount of the coating antigen was 1. Mu.g/ml, 100. Mu.l/well; the coating buffer was PBS (pH 7.4); the secondary antibody is goat anti-mouse IgG marked by HRP; the absorbance at 450nm was measured using a microplate reader with 630nm as the reference wavelength.

TABLE 2 ELISA detection results of hybridoma cell supernatants

4. Antibody production

Performing one round of subcloning on the 10 hybridoma cells screened in the step 3, performing small-scale antibody production by using a rotary bottle culture or mouse ascites mode, performing affinity purification by using a protein A/G column, and storing the purified antibody in Phosphate Buffer (PBS) by using a dialysis method.

5. Antibody pairing experiments

The 10 antibodies were labeled with Biotin (Biotin), the porous plate was coated with unlabeled antibody (capture antibody), the Biotin-labeled antibody was used as a detection antibody, the antigen rhFVIIa-Fc fusion protein was subjected to double dilution, and the 10 antibodies were combined and subjected to sandwich ELISA detection. The method comprises the following specific steps:

(1) Coating: 5G1H5, 8F10C9, 12E1B6, 13G7B9, 18B10E9 to 2.5ug/ml,100 ul/well coating, film coating, overnight at 4 ℃.

(2) Washing the plate: plates were washed with PBST (Tween 20 content 0.05%), 350 ul/well, left to stand, and the plate washed repeatedly a total of 4 times.

(3) Closing: blocking with 3% BSA (BSA dilution with PBST), 350 ul/well, membrane coating, and standing at 37℃for 2h.

(4) Washing the plate: plates were washed with PBST, 350 ul/well, left to stand, and the plate washed repeatedly a total of 4 times.

(5) Adding a sample: the rhFVIIa-Fc fusion protein was taken, diluted to 100ng/ml with a gradient of PBST containing 1% BSA, followed by 3-fold dilution, 5 gradients total, 1% BSA as blank, 2 wells each, and incubation at 37℃for 1h after membrane coating.

(6) Washing the plate: plates were washed with PBST, 350 ul/well, left to stand for 1, and the plate washing repeated 4 times.

(7) Adding biotinylated antibody: biotinylated 5G1H5, 8F10C9, 12E1B6, 13G7B9, 18B10E9 antibodies were diluted to 1ug/ml with 1% BSA, 100 ul/well and left to stand at 37℃for 1H after coating.

(8) Washing the plate: plates were washed with PBST, 350 ul/well, left to stand, and the plate washed repeatedly a total of 4 times.

(9) Adding avidin HRP antibody: with 1% bsa1: streptavidin (HRP) (Abcam, ab 7403) antibodies were diluted 10000 times, 100 ul/well, and left to stand at 37℃for 1h after coating.

(10) Washing the plate: plates were washed with PBST, 350 ul/well, left to stand, and the plate washed repeatedly a total of 4 times.

(11) Color development and termination: mu.l of TMB developing solution (Soxhaust, PR 1200) was added to each well, and the mixture was developed at 37℃for 5 minutes in an electrically heated incubator. The reaction was stopped by adding 50. Mu.l of stop solution (2M sulfuric acid) to each well.

(12) And (3) detection: the absorbance at 450nm was measured using a microplate reader with 630nm as the reference wavelength.

TABLE 3 antibody pairing results

( And (3) injection: the antibodies herein are numbered identically to the corresponding hybridoma cells )

As can be seen from table 3, the other paired antibodies showed good effects, except that 13G7B9 and 18B10E9 may have competing binding. The 13G7B9 and 12E1B6-Biotin antibody pairs were selected for use in establishing subsequent detection methods.

By sequencing, antibody 13G7B9 has a heavy chain shown as SEQ ID NO. 17, and a light chain shown as SEQ ID NO. 18; antibody 12E1B6 has a heavy chain shown as SEQ ID NO. 19, and a light chain shown as SEQ ID NO. 20.

Antibody 13G7B9:

antibody 12E1B6:

Example 2 biological analysis method for detecting drug concentration in cynomolgus monkey plasma

The embodiment establishes an ELISA method for quantitatively detecting the concentration of the rhFVIIa-Fc fusion protein in the blood plasma of the cynomolgus monkey, and performs partial methodological verification on the aspects of standard curve, quantitative range, accuracy, precision and durability of the method, thereby providing arguments for biological analysis of toxicology and drug generation tests.

1. Principle of the method

The 13G7B9 antibody is used as a capture reagent to be coated on an ELISA plate, and rhFVIIa-Fc fusion proteins in standard curve Samples (STDs), quality Control Samples (QCs), verification test samples (VSs) and samples to be tested are captured on the ELISA plate. After removal of unbound material by plate washing, the antibody 12E1B6-Biotin was used as detection reagent for specific detection of captured rhFVIIa-Fc fusion protein. After washing, peroxidase-coupled streptavidin was added to bind 12E1B6-Biotin, and TMB was added as a substrate to the ELISA plate after washing again. TMB and peroxide in the substrate solution under the action of HRP produce a colorimetrically detectable signal proportional to the concentration of rhFVIIa-Fc fusion protein in the sample. After termination of the reaction by addition of 1M HCl, the absorbance (OD) was measured at 450nm (reference wavelength 630 nm). OD values of QCs, VSs and unknown samples can be converted into concentration values by regression according to the relation between the signal and the concentration values of the standard curve, and the regression calculation is performed on the authenticated Watson LIMS ^TM Regression models built with a 5-logical parameter weighting factor of 1/Y2 in (Thermo Fisher Scientific inc., PA) or SoftMax Pro GxP.

2. Key reagents and major instrumentation

Antibodies 13G7B9 and 12E1B6-Biotin, peroxidase-conjugated streptavidin (SA-HRP, available from Jackson Immuno Research, 016-030-084) prepared in example 1; the microplate reader Molecular Devices, versamax/SpectraMax M3/SpectraMax M5/SpectraMax iD3, carries softMax Pro GxP 7.0.3/7.0.3SP1.

3. Preparation of standard curve and quality control sample

TABLE 4 preparation of standard curve samples

TABLE 5 preparation of quality control samples

4. Experimental procedure

1) Preparing a coating working solution: 13G7B9 was diluted with 1 XPBS buffer to a final concentration of 1. Mu.g/mL.

2) Coating: adding 100 mu L/hole of coating working solution into a 96-well plate, covering a sealing plate membrane, and placing the plate membrane at 2-8 ℃ for incubation for 16-18 h.

3) Washing the plate: the liquid in the 96-well plate is taken out, the plate is washed 3 to 5 times by 300 mu L/Kong Xiye, and the 96-well plate is patted dry on clean absorbent paper.

4) Closing: adding 300 mu L/well of blocking solution (1% BSA-PBST) into a 96-well plate, covering a sealing plate membrane, standing at room temperature for incubation for 1.5-2.5 h, and preserving for 7 days at 2-8 ℃ after blocking.

5) MRD treatment: samples were added to the dilution wells at 8. Mu.L/Kong Shanlie each, then 232. Mu.L assay buffer (1% BSA-PBST) was added to the corresponding dilution wells, and the covered plate membrane was placed on a microplate shaker at room temperature at 500rpm for no less than 10 minutes (the volume could be increased or decreased proportionately).

6) Washing the plate: the wells were removed and the plates were washed 3-5 times with 300. Mu.L/Kong Xiye (PBST) and the wells were dried on clean absorbent paper. If not used for the test on the same day, the ELISA plate can be stored at-15 ℃ to-30 ℃ for 1 month.

7) Sample adding: 100 mu L/well of MRD treated sample is added into a 96-well plate, covered by a sealing plate membrane and placed at room temperature of 150-300 rpm for incubation for 55-65 min.

8) Preparing a detection working solution: detecting a working solution I: diluting 12E1B6-Biotin with assay buffer 1000-fold; detecting a working solution II: SA-HRP stock was diluted 200000-fold with assay buffer.

9) Washing the plate: the liquid in the 96-well plate is taken out, the plate is washed 3 to 5 times by 300 mu L/Kong Xiye, and the 96-well plate is patted dry on clean absorbent paper.

10 Adding detection working solution I: and adding 100 mu L/hole of detection working solution I into the 96-well plate, covering a sealing plate membrane, and placing the sealing plate membrane at the room temperature of 150-300 rpm for incubation for 55-65 min.

11 Plate washing: the liquid in the 96-well plate is taken out, the plate is washed 3 to 5 times by 300 mu L/Kong Xiye, and the 96-well plate is patted dry on clean absorbent paper.

12 Adding detection working solution II: and adding 100 mu L/hole of detection working solution II into the 96-well plate, covering a sealing plate membrane, and placing the sealing plate membrane at the room temperature of 150-300 rpm for incubation for 55-65 min.

13 Preparing TMB working solution: and fully mixing TMB color development liquid A and TMB color development liquid B in a volume ratio of 1:1.

14 Plate washing: the liquid in the 96-well plate is taken out, the plate is washed 3 to 5 times by 300 mu L/Kong Xiye, and the 96-well plate is patted dry on clean absorbent paper.

15 Color development): 100 mu L/hole TMB working solution is added into the 96-well plate, and the mixture is kept stand and incubated for 6 to 10 minutes at room temperature in a dark place.

16 Terminating: 50. Mu.L/well of stop solution was added to the 96-well plate, and the edges of the 96-well plate were gently tapped for mixing.

17 Reading a plate: the 96-well plate is put into an enzyme labeling instrument, the detection wavelength is 450nm, and the reference wavelength is 630 nm.

5. Methodological validation results

(1) Precision and accuracy

Evaluation of precision and accuracy 6 successful accuracy and precision analysis batch experiments were completed on 2 working days by 2 analysts, each analysis batch containing one set of standard curves, 1 blank control sample and 3 sets of quality control samples, each set of quality control samples comprising five levels: LLOQ, LQC, MQC, HQC and ULOQ.

TABLE 6 quality control of the accuracy, precision and total error data statistics between samples

(2) Standard curve, quantitative range and sensitivity

The results show that the measurement value batch-to-batch precision% CV of the standard curve points (except anchor points) of the rhFVIIa-Fc fusion protein in the blood plasma of the cynomolgus monkey is less than or equal to 2.53%, the quantitative range is 50.00 ng/mL-3200.00 ng/mL, and the sensitivity of the method is 50.00ng/mL.

(3) Robustness and repeatability

Robustness refers to the ability of an analytical method to be unaffected when there is a small change in the parameters of the method (incubation time, coating time, etc.), mainly verifying the reliability and stability of the method. Run 1 in the accuracy and precision test was evaluated using the shortest reaction time in the table below and Run 3 using the longest reaction time in the table below. The data show that the variation in reaction time in the following table has no significant effect on the results of this test.

TABLE 7 investigation of robustness and repeatability

Robustness and repeatability are assessed by accuracy and precision tests. The method performed 6 successful accuracy and precision tests, including a robustness verification test, so the test method was stable and repeatable. The results meet the requirements of the test scheme.

(4) Selectivity of

rhFVIIa-Fc fusion protein standard (supplied by Cheshi, zhengzhou, lot number 109202105P 01R) was added to 10 different individual cynomolgus monkey plasma samples and 3 different individual hemolyzed cynomolgus monkey plasma samples with a whole blood content of 2% (V whole blood/V total volume) (supplied by Jiangsu dingtai, acquisition table number 20210812.1, anticoagulant sodium citrate) to prepare 3200.00ng/mL (ULOQ), 50.00ng/mL (LLOQ), 0.00ng/mL of selective samples, and tested.

The selective verification result shows that: the measurement values of blank cynomolgus monkey plasma samples without obvious hemolysis and without added standard substances from 10 different individual sources are all lower than the lower limit of quantification; samples added to both LLOQ (50.00 ng/mL) and ULOQ (3200.00 ng/mL) levels were validated. Wherein the accuracy of the sample added with the standard substance to the LLOQ level is between 3.81 and 20.11 percent, and the precision is between 0.35 and 7.11 percent; the accuracy of the standard substance added to the ULOQ level sample is between 0.78 and 8.00 percent, and the precision is between 0.08 and 2.49 percent.

The hemolysis selectivity verification result shows that: whole blood from 3 different individual sources without added standard was below the lower limit of quantitation in 2% hemolyzed cynomolgus monkey plasma samples, and samples added to levels of LLOQ (50.00 ng/mL) and ULOQ (3200.00 ng/mL) were validated. Wherein the accuracy of the standard substance added to the LLOQ level sample is between-3.57 percent and 1.41 percent, and the precision is between 1.26 percent and 5.61 percent; the accuracy of the sample added with the standard substance to the ULOQ level is between-15.11% and-11.64%, and the precision is between 0.54% and 1.93%.

All the results meet the acceptance criteria of the test scheme.

(5) Parallelism of

The accuracy was determined by diluting 3 high-concentration real samples 500, 200, 100-fold with a blank matrix, respectively. The results show that: the precision of final concentration calculated after 3 real samples are diluted by 3 different times is between 7.02 percent and 15.60 percent, and the parallelism acceptance standard is met.

(6) Dilution linearity and hook effect

Dilution linearity validation 200, 1000, 2000, 20000 dilutions, 3 replicates per dilution validation sample.

And (3) data display: the accuracy of the diluted linear sample and the precision of the complex pore signal value meet the acceptance standard, which shows that 200 times, 1000 times and 2000 times dilution of the plasma sample containing the high-concentration rhFVIIa-Fc fusion protein reference substance can not influence the accuracy of sample measurement.

The hook effect was mainly verified at 380000.00, 38000.00ng/mL, and the verification samples at each concentration level were subjected to multiplex well analysis.

And (3) data display: no hook effect was detected for samples up to 380000.00 ng/mL.

(7) Specificity (specificity)

Quality control samples of HQC and LQC with 2 times concentration and Human IgG (purchased from Absin, cat. No. abs 20037) with different concentrations are respectively prepared from cynomolgus monkey plasma as interference samples, and then the quality control samples are respectively mixed with the interference samples with different concentrations (60000.00, 6000.00, 600.00 ng/mL) in equal volumes to prepare a specificity verification sample.

The results show that: the accuracy of the specificity verification sample and the precision of the complex pore signal value meet the acceptance standard, which shows that the existence of the Human IgG interferent does not interfere with the measurement of the rhFVIIa-Fc fusion protein.

(8) Stability of

In the stability detection test of the quality control sample, the quality control sample with two concentrations of HQC (2400.00 ng/mL) and LQC (150.00 ng/mL) is selected for the investigation of the freezing and thawing stability (-65 ℃ to-90 ℃ per room temperature) and the long-term storage stability at room temperature, 2 ℃ to 8 ℃, 15 ℃ to 30 ℃ below zero and 3 times/6 times. The calculated concentration of each concentration stability sample is within% Bias plus or minus 20.00%, and the complex pore signal value% CV is not more than 20.00%.

The results show that: the quality control samples are respectively stored at room temperature for 24h and at the temperature of 2-8 ℃ for 24h, stored at the temperature of minus 15-minus 30 ℃ for 9d 16h, frozen and thawed for 3 times/6 times at the room temperature of minus 65-minus 90 ℃ and stored at the temperature of minus 65-minus 90 ℃ for 78 days, and the detection results meet the acceptance standard.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the scope of the present application. Any modifications, equivalent substitutions, improvements, etc. that are within the spirit and principles of the present application are intended to be included within the scope of the present application.

Claims (10)

1. An antibody or antigen-binding fragment thereof that binds a recombinant human factor VIIa-Fc fusion protein comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; wherein, the liquid crystal display device comprises a liquid crystal display device,

the HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6; or alternatively

The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising:

a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 13 and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 14; or (b)

A heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 15, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 16.

3. The antibody or antigen-binding fragment thereof of claim 1, comprising:

A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 13, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 14; or (b)

A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 15, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 16.

4. The antibody or antigen-binding fragment thereof of claim 1, comprising:

a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 17 and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 18; or (b)

A heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 19, and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 20.

5. The antibody or antigen-binding fragment thereof of claim 1, comprising:

A heavy chain with an amino acid sequence shown as SEQ ID NO. 17, and a light chain with an amino acid sequence shown as SEQ ID NO. 18; or (b)

A heavy chain with an amino acid sequence shown as SEQ ID NO. 19, and a light chain with an amino acid sequence shown as SEQ ID NO. 20.

6. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-5; or, a recombinant vector comprising said polynucleotide; or, a host cell comprising said polynucleotide or recombinant vector; or a hybridoma cell for expressing the antibody or antigen-binding fragment thereof of any one of claims 1-5.

7. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-5 for detecting the presence or amount of recombinant human factor vila-Fc fusion protein in a sample.

8. A method for detecting the presence or amount of recombinant human factor vila-Fc fusion protein in a sample using the antibody or antigen binding fragment thereof of any one of claims 1-5, wherein the method is a sandwich ELISA method, which antibody or antigen binding fragment thereof serves as a capture antibody and/or detection antibody.

9. An antibody combination for detecting a recombinant human factor vila-Fc fusion protein comprising two of the antibodies or antigen-binding fragments thereof of any one of claims 1-5.

10. A kit for detecting a recombinant human factor vila-Fc fusion protein comprising at least one of the antibodies or antigen-binding fragments thereof of any one of claims 1-5.