CN118344476A - Human C reactive protein monoclonal antibody, antibody pair, and detection reagent or kit and application thereof - Google Patents
Human C reactive protein monoclonal antibody, antibody pair, and detection reagent or kit and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of antibody preparation, and particularly relates to a human C-reactive protein monoclonal antibody, an antibody pair, a detection reagent or a kit and application thereof. The monoclonal antibody is a first antibody or a second antibody, the amino acid sequences of CDR1-3 on the light chain variable region of the first antibody are respectively shown as SEQ ID NO.5-7, and the amino acid sequences of CDR1-3 on the heavy chain variable region are respectively shown as SEQ ID NO. 8-10; the amino acid sequences of CDR1-3 on the light chain variable region of the second antibody are shown as SEQ ID NO.15-17, and the amino acid sequences of CDR1-3 on the heavy chain variable region are shown as SEQ ID NO. 18-20. The antibody and the antibody have the advantages of strong specificity and high affinity for human CRP protein, and are sensitive, accurate, specific, stable, strong in anti-interference performance and the like when being used for detecting the human CRP protein.
Description
Technical Field
The invention relates to the technical field of antibody preparation, in particular to a human C-reactive protein monoclonal antibody, an antibody pair, a detection reagent or a kit and application thereof.
Background
C-reactive protein (CRP) is an acute phase reaction protein, and is mainly synthesized in the liver and then exists in human blood after being induced by cytokines such as IL-6, IL-2, TNF and the like when tissues are damaged, inflamed or infected, and CRP can be generated in other local tissues such as nerve cells, monocytes, lymphocytes and atherosclerosis plaques in a small amount, the half-life of the CRP in the blood is stable for about 19 hours, and the CRP concentration in the blood mainly depends on the generation amount of the liver. The human CRP gene is located on human chromosome 1, between 1q21 and 1q23, contains 2263 nucleotides, and has an intron. CRP protein forms a cyclic pentamer by 5 identical subunits, each monomer is in a sphere shape (composed of 206 amino acids), forms a symmetrical cyclic pentasphere in a non-covalent form, is a member of a pentameric protein (Pentagin) protein family, has a relative molecular weight of 115-140KD, a sedimentation coefficient of 6.5-7.5, an isoelectric point of 5.5 and is not easily dissolved in water.
CRP has a variety of biological functions and is involved in a variety of own physiological and pathological processes. CRP has a high affinity for phosphatidylcholine residues and can bind to a variety of self-ligands (e.g., plasma cell lipoproteins, cell membrane damaging cells, small ribosomal protein particles, opsonin cells, etc.) or exotic ligands (e.g., polysaccharides, phospholipids, and components of microorganisms such as bacteria, fungi, parasites, etc.). After CRP binds to these ligands, it activates the primary stages of the classical pathway of complement activation, limiting the development and intensity of the late inflammatory response of complement activation. CRP can also increase lymphocyte activity and strengthen phagocytic capacity of phagocytes, thereby eliminating pathogenic microorganisms invading the organism and tissue cells damaged, necrotic and apoptotic, and playing an important role in protecting the natural immune process of the organism. CRP is widely involved in various inflammatory and immune reactions in the body. CRP content in serum of healthy people is extremely small, concentration is 0.07-8.0mg/L, concentration is obviously increased after infection occurs, the concentration starts to increase after 6-8 hours, peak value is reached after 24-48 hours, the concentration can be increased to 1000-2000 times of normal value, and the increasing amplitude is in direct proportion to the infection degree; and the concentration is rapidly reduced after the inflammation is cured, and the normal level can be recovered within one week. The CRP changes independently of the individual differences, body status and therapeutic drugs of the patient, are non-specific inflammatory response markers, and can be at levels that reflect the potential inflammatory activity and severity of the body. At present, the content variation plays an important role in a plurality of fields: 1) Diagnosis and differential diagnosis of bacterial or viral infections. Studies show that CRP is 10-50mg/L and is low-grade inflammation; 50mg/L, a bacterial infection can be determined; 100mg/L, which indicates a serious disease process and often indicates the presence of a bacterial infection. 2) Auxiliary diagnosis of neonatal infectious diseases. Neonatal CRP is produced by the fetal stage itself, and delivery causes CRP to be stressed, and CRP is not specifically measured in 3d after birth. Most bacterial infections have elevated CRP in newborns over a period of time. CRP is a means of diagnosing neonatal sepsis. Elevation of CRP can also occur in the case of non-bacterial infections such as meconium aspiration, respiratory distress syndrome, and the like. 3) Monitor the change of illness and postoperative infection. When the operation is accompanied by bacterial infection, high-value CRP can continuously exist, and the concentration of the CRP is 250-350mg/L after normal operation for 2-3 d; the post-operation time is reduced to below 30 mg/L. 4) And observing the application curative effect of the antibiotics. CRP can be used as a safe and reliable index for stopping the antibiotic of bacterial infection, guiding the application of the antibiotic, evaluating the curative effect of the antibiotic, guiding the treatment course and the like. In addition, CRP has a great correlation with the occurrence and development of some infectious diseases, cardiovascular diseases, autoimmune diseases, malignant tumors, depression and the like.
In view of the important biological functions of CRP, detecting this index becomes particularly important. Currently, immunological methods such as enzyme-linked immunosorbent assay, immunoturbidimetry, immunoprecipitation and immunofluorescence assay for detecting CRP have become the mainstream direction because of simple operation and easy determination of results. Different methods all require specific monoclonal antibodies directed against CRP. The murine monoclonal antibodies are used in traditional clinical diagnosis, and have low affinity and specificity, and can detect CRP of more than 8mg/L in infection or tissue injury, but cannot detect CRP level change at low concentration (< 8 mg/L). Therefore, the development of novel high affinity antibodies that bind efficiently and specifically to CRP is of great significance and clinical value.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a rabbit monoclonal antibody and an antibody pair with strong specificity and high affinity for resisting human CRP protein, the detection system constructed by the antibody and the antibody pair is used for detecting human CRP protein, has the advantages of sensitivity, accuracy, specificity, stability, strong anti-interference performance and the like, and is suitable for qualitatively and quantitatively detecting human CRP protein with very small amount, thereby the invention also provides application of the antibody and the antibody pair in preparing human CRP protein detection reagent or kit, and provides detection reagent or kit containing the antibody and the antibody pair.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
The first aspect of the present invention provides a monoclonal antibody of human C-reactive protein, which is a first antibody or a second antibody, wherein: the amino acid sequences of CDR1-3 on the light chain variable region of the first antibody are respectively shown in SEQ ID NO.5-7, and the amino acid sequences of CDR1-3 on the heavy chain variable region are respectively shown in SEQ ID NO. 8-10; the amino acid sequences of CDR1-3 on the light chain variable region of the second antibody are shown as SEQ ID NO.15-17, and the amino acid sequences of CDR1-3 on the heavy chain variable region are shown as SEQ ID NO. 18-20.
Further, the amino acid sequence of the first antibody light chain variable region is shown as SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4; the amino acid sequence of the light chain variable region of the second antibody is shown as SEQ ID NO.13, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 14.
Further, the amino acid sequence of the first antibody light chain is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 2; the amino acid sequence of the light chain of the second antibody is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 12.
Further, the first antibody or the second antibody is a full-length antibody or an antibody fragment; the antibody fragment is selected from at least one of Fab fragment, F (ab) 2 fragment, fv fragment, (Fv) 2 fragment, scFv fragment and sc (Fv) 2 fragment.
In a second aspect, the invention provides a nucleic acid molecule encoding a first antibody or a second antibody as described above, a recombinant vector or a host cell comprising said nucleic acid molecule.
In a third aspect, the invention provides a human C-reactive protein antibody pair, consisting of a first antibody and a second antibody as described above.
In a fourth aspect, the invention provides the use of a human C-reactive protein monoclonal antibody or antibody pair as described above in the preparation of a human C-reactive protein detection reagent or kit.
In a fifth aspect the present invention provides a human C-reactive protein detection reagent or kit comprising a primary antibody and/or a secondary antibody as described above.
Further, the detection reagent or kit comprises a first antibody and a second antibody, wherein the first antibody is a capture antibody, the second antibody is a detection antibody, and the second antibody is modified with a detectable label.
Still further, the detectable label is biotin.
Compared with the prior art, the invention has the advantages and positive effects that:
The antibody specificity recognition human CRP protein containing the CDR sequence provided by the invention has the advantages of good binding activity, high affinity and the like, and the first antibody and the second antibody specifically recognize different antigenic determinants of the human CRP protein, can form a double-antibody sandwich ELISA detection system, is beneficial to realizing the sensitivity, accuracy, specificity and stability detection of trace-level human CRP protein, and has important significance and wide application prospect in the fields of clinical diagnosis and scientific research.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings that are required to be used in the description of the embodiments will be briefly described below.
FIG. 1 is a diagram of a vector used for constructing a rabbit monoclonal antibody expression vector according to example 1 of the present invention, from left to right, pRB322 vector map carrying a light chain constant region and a heavy chain constant region, respectively;
FIG. 2 is a graph showing the affinity of monoclonal antibody A of example 2 of the present invention for binding to human CRP protein;
FIG. 3 is a graph showing the affinity of monoclonal antibody B of example 2 of the present invention for binding to human CRP protein;
FIG. 4 is a standard curve of the detection of human CRP protein based on the double-antibody sandwich ELISA system established by monoclonal antibody A and monoclonal antibody B in example 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. The examples described herein are intended to illustrate the invention only and are not intended to limit the invention.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit or scope of the appended claims. It is to be understood that the scope of the invention is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.
For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
In addition, it is noted that unless otherwise defined, in the context of the present invention, scientific and technical terms used should have meanings commonly understood by one of ordinary skill in the art.
The terms "comprising," "including," "having," and the like are intended to be non-limiting, as other steps and other ingredients not affecting the result may be added. The term "and/or" should be taken to refer to a specific disclosure of each of the two specified features or components with or without the other. For example, "a and/or B" is considered to include the following: (i) A, (ii) B, and (iii) A and B. The terms "first," "second," and the like, are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order, it being understood that such uses may be interchanged where appropriate.
The terms "rabbit monoclonal antibody", "antibody" and "mab" and the like have the same meaning and are used interchangeably, and refer to antibodies that specifically bind to human C-reactive protein unless otherwise specified. The terms "Human C-reactive protein", "Human CRP" and the like have the same meaning and are used interchangeably. The modifier "rabbit" means that the complementarity determining regions of the antibody are derived from rabbit immunoglobulin sequences.
An antibody is an immunoglobulin molecule capable of specifically binding to an antigen or epitope of interest through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" is to be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full length antibodies, antibody fragments, and genetic or chemical modifications thereof, as long as they exhibit the desired antigen binding activity. An antibody fragment may be one or more portions or fragments of a full-length antibody that retains the ability of the antibody to specifically bind to an antigen of interest. In typical examples, the antibody fragments include: fab, fab', F (ab) 2、F(ab')2、Fv、(Fv)2、scFv、sc(Fv)2.
A typical antibody molecule (full length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be divided into two types, kappa and lambda chains, respectively; heavy chains can be categorized into five, μ, δ, γ, α and ε chains, respectively, and antibodies are defined as IgM, igD, igG, igA and IgE, respectively. The amino acid sequences of the heavy and light chains near the N-terminus vary greatly, the other portions of the amino acid sequences are relatively constant, the region of the light and heavy chains near the N-terminus, where the amino acid sequences vary greatly, is referred to as the variable region (V), and the region near the C-terminus, where the amino acid sequences are relatively stable, is referred to as the constant region (C). Heavy chain variable regions (VH) and light chain variable regions (VL) are typically the most variable parts of antibodies and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (hypervariable region, HVR) also known as Complementarity Determining Regions (CDRs) which are circular structures, and Framework Regions (FR) where the heavy and light chain CDRs are held closely together and cooperate with one another by the FR regions to form surfaces complementary to the three-dimensional structure of the antigen or epitope of interest, determining the specificity of the antibody, and are the sites for antibody recognition and binding to the antigen. The FR region is the more conserved part of VH and VL, which are generally in the β -sheet configuration, joined by three CDRs forming a connecting loop. Each VH and VL is typically composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
CDRs and FR can be identified according to Kabat definition, chothia definition, a combination of both Kabat definition and Chothia definition, abM definition, contact definition, IMGT unique number definition and/or conformational definition, or any CDR determination method known in the art. As used herein, is defined by the Kabat numbering system.
The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of an antibody. CL lengths of different classes of igs (κ or λ) are substantially identical, but CH lengths of different classes of igs are different, e.g. IgG, igA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4. The amino acid sequences of the antibody heavy and light chain constant regions are well known in the art.
Full length antibodies are the most complete antibody molecular structure, having a typical Y-type molecular structure, and thus, "full length antibodies", "complete antibodies" and "Y-type antibodies" are used interchangeably in the context of the present invention.
The term "Antigen binding fragment (Fab)" is a region of an antibody molecule that binds Antigen and consists of the complete light chain (variable and constant regions) and part of the heavy chain (variable and one constant region fragment), whereby fragments such as Fab, F (ab ') 2, fab' can be obtained by proteolytic cleavage of the full-length antibody. For example, igG can be degraded into two Fab fragments and one Fc fragment by papain; igG can be degraded into a F (ab ') 2 fragment and a pFC' fragment by pepsin. The F (ab ') 2 fragment was further reduced to form two Fab' fragments. Because the Fab has an antigen binding region and a partial constant region, the Fab not only has antibody-antigen affinity like a single chain antibody (scFv), excellent tissue penetrating power and the like, but also has a more stable structure, and is widely applied to clinical diagnosis and treatment.
The term "variable fragment (Fv)" is located at the N-terminus of an antibody Fab fragment, contains only the variable region, and consists of one variable region of one light chain and one heavy chain, is a dimer of one VH and one VL that are non-covalently bound (VH-VL dimer), and the 3 CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer, with the ability to recognize and bind antigen, although with less avidity than an intact antibody.
The term "Single-chain antibody (scFv)" refers to a minimum antibody fragment in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked by a flexible linker (linker, typically consisting of 10 to 25 amino acids), which retains the binding specificity of the original antibody to an antigen, and the linker in the present invention is not particularly limited as long as it does not interfere with the expression of the antibody variable regions linked at both ends thereof. Compared with full-length antibodies, scFv has the characteristic of small molecular weight, thus having higher penetrability and lower immune side reaction.
In some embodiments, the full length sequence of an antibody or antibody fragment of the invention may comprise Complementarity Determining Regions (CDRs) and Framework Regions (FRs) from a rabbit immunoglobulin sequence. In other embodiments, the antibodies may comprise amino acid residues encoded by non-rabbit immunoglobulin sequences, e.g., humanized antibodies, chimeric antibodies, etc., to reduce body rejection while maintaining the desired specificity, affinity. The term "chimeric antibody" refers to an antibody in which a portion is derived from a particular source or species, while the remainder is derived from a different source or species. The term "humanized antibody" is a chimeric antibody in which the CDR regions of a non-human antibody, such as a rabbit antibody, and the FR regions derived from a human, in some cases, the variable regions of a non-human antibody bind to the constant regions of a human antibody, e.g., a human rabbit chimeric antibody; in other cases, the CDR regions of a non-human antibody bind to FR regions and constant regions derived from human antibody sequences, i.e., the CDR regions of a non-human antibody are grafted onto human antibody Framework (FR) sequences derived from single or multiple other human antibody variable region framework sequences. In the present invention, the CDR regions in the chimeric or humanized antibody are derived from rabbit-derived CDR regions.
The terms "monoclonal antibody" or "mab" and the like are used interchangeably and refer to a homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for small amounts of mutations and/or post-translational modifications (e.g., isomerization, amidation) that may occur naturally. "monoclonal antibodies" are highly specific, exhibiting a single binding specificity and affinity for the same or substantially the same epitope on an antigen. The modifier "monoclonal" indicates the antibody is obtained from a substantially homogeneous population of antibodies and is not to be construed as limiting the source or manner of preparation of the antibody. The antibodies can be prepared by a variety of methods including, but not limited to, hybridoma methods, phage display methods, yeast display methods, recombinant DNA methods, single cell screening, or single cell sequencing methods.
The term "specific binding" is a term well known in the art that exhibits "specific binding," "specific binding," or is referred to as "preferential binding" if a molecule reacts more frequently, more rapidly, longer in duration, and/or with greater affinity to a particular antigen or epitope of interest than to other antigens or epitopes of interest, and does not necessarily require (although may include) exclusive binding.
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
The embodiment of the invention provides a human C-reactive protein monoclonal antibody, which is a first antibody or a second antibody, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region and the heavy chain variable region comprise 3 Complementarity Determining Regions (CDRs) respectively named as CDR1, CDR2 and CDR3; wherein: the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the first antibody are respectively shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are respectively shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10; the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the second antibody are shown as SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17 respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20 respectively.
The antibody specificity recognition human CRP protein containing the CDR sequence provided by the invention has the advantages of good binding activity, high affinity and the like, the affinity constant K D of the first antibody and the human CRP protein is 1.49 multiplied by 10 -9 M, the affinity constant K D of the second antibody and the human CRP protein is 2.88 multiplied by 10 -10 M, and the two antibodies can recognize different antigenic determinants on the surface of the CRP protein and can be used for developing a double antibody sandwich method enzyme-linked immunosorbent (ELISA) detection system; the double-antibody sandwich ELISA system is constructed by taking the first antibody as a capture antibody and the second antibody as a detection antibody, CRP with different concentrations is quantitatively detected, the linear range (31.2-2000 pg/mL) is wide, the detection limit is as low as 25.97pg/mL, and the detection specificity and the detection sensitivity are good; the invention provides a solution way for stably detecting the trace level of human CRP protein in samples such as serum, cell culture solution and the like, and has important significance and wide application prospect in the fields of clinical diagnosis and scientific research.
Optionally, the light chain variable region and the heavy chain variable region of the monoclonal antibody of the present invention further comprise 4 Framework Regions (FR), and 4 FR and 3 CDR are sequentially staggered in each chain to constitute the variable region. Specifically, the amino acid sequence of the light chain variable region (VL) of the first antibody is shown as SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO. 4. The amino acid sequence of the light chain variable region (VL) of the second antibody is shown as SEQ ID NO.13, and the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO. 14.
Optionally, the rabbit monoclonal antibodies of the invention further comprise a light chain constant region and a heavy chain constant region, CL and VL comprising the light chain and CH and VH comprising the heavy chain. The constant regions of antibodies are typically obtained by public interrogation, such as: the rabbit source IGG GAMMA C REIGN was searched for CH and the rabbit source IGG KAPPA C REIGN was searched for CL via IMGT online database (www.imgt.org). Specifically, the amino acid sequence of the light chain (FL chain) of the first antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain (FH chain) is shown as SEQ ID NO. 2. The amino acid sequence of the light chain (FL chain) of the second antibody is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain (FH chain) is shown as SEQ ID NO. 12. Wherein the light chain constant region of the first antibody and the second antibody is a kappa chain and the heavy chain constant region is of the IgG1 type.
It should be noted that the monoclonal antibody of the present invention may be a full-length antibody (having a typical Y-type molecular structure) or an antibody fragment, which refers to a polypeptide that retains substantially the same biological function or activity as the full-length form of a rabbit monoclonal antibody, specifically, an antibody fragment includes CDR regions as described above, more preferably has variable regions as described above, thereby retaining intact CRP antigen recognition and binding sites capable of binding to the same antigen, particularly to the same epitope, as the full-length antibody.
Such antibody fragments include, but are not limited to: (i) A Fab fragment, a monovalent fragment consisting of the variable region and the first constant region of each heavy and light chain; (ii) A F (ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fv fragment consisting of one heavy chain variable region and one light chain variable region of an antibody; (iv) (Fv) 2 fragment consisting of two Fv fragments covalently linked together; (v) An scFv fragment, an Fv fragment consisting of a single polypeptide chain, a heavy chain variable region and a light chain variable region joined by a linker; (vi) The sc (Fv) 2 fragment is obtained by ligating two heavy chain variable regions and two light chain variable regions via a linker or the like. These antibody fragments can be obtained by conventional techniques in the art.
Yet another embodiment of the invention provides a nucleic acid molecule encoding a first antibody and/or a second antibody as described above, a recombinant vector or a host cell comprising said nucleic acid molecule.
The nucleic acid molecule may be in the form of DNA (e.g., cDNA or genomic DNA or synthetic DNA) or RNA (e.g., mRNA or synthetic RNA). The DNA may be single-stranded or double-stranded, or may be a coding strand or a non-coding strand.
The sequence of the nucleic acid molecule is deduced by conventional means such as codon encoding rules according to the amino acid sequence of the antibody. The full-length sequence of the nucleic acid molecule or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis. The obtained nucleic acid molecules are inserted into an expression vector, then host cells are transfected, and the transfected host cells are cultured under specific conditions, so that the antibody of the invention can be expressed and obtained.
The original vector from which the recombinant vector is constructed is a variety of vectors conventional in the art, as long as it is capable of harboring the nucleic acid molecule. Typical vectors include plasmids, viral vectors, phages, cosmids and minichromosomes. Plasmids are the most common form of vector, and thus, in the context of the present invention, vectors are used interchangeably with plasmids. The vector may be a cloning vector (i.e., for transferring the nucleic acid molecule into a host and for mass propagation in a host cell) or an expression vector (i.e., comprising the necessary genetic elements to allow expression of the nucleic acid molecule inserted into the vector in a host cell). The nucleic acid molecules of the invention may be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecule. This is a well known technique and will not be described in detail here.
Nucleic acid molecules encoding the heavy and light chains of the antibodies of the invention may be constructed separately on two vectors, which may be introduced into the same or different host cells. When the heavy and light chains are expressed in different host cells, each chain may be isolated from the host cell in which it is expressed and the isolated heavy and light chains mixed and incubated under appropriate conditions to form the antibody. In other embodiments, the nucleic acid molecules encoding the heavy and light chains of the rabbit monoclonal antibodies of the invention may also be cloned into a vector, each nucleic acid sequence being linked downstream of a suitable promoter; for example, each nucleic acid sequence encoding a heavy chain and a light chain may be operably linked to a different promoter, or the nucleic acid sequences encoding the heavy chain and the light chain may be operably linked to a single promoter such that both the heavy chain and the light chain are expressed from the same promoter. The choice of expression vector/promoter depends on the type of host cell used to produce the antibody.
Transfection or transformation of the recombinant vector into a host cell according to the present invention is carried out using conventional techniques. When the host is a prokaryote such as E.coli, competent cells capable of absorbing DNA are obtained after the exponential growth phase and treated with CaCl 2 or MgCl 2; or by microinjection, electroporation, or liposome encapsulation. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging, and the like. The host cell may be a prokaryotic or eukaryotic cell. Representative examples are: coli, streptomycete, salmonella typhimurium, yeast, drosophila S2 or Sf9 cells, mammalian CHO, COS7, 293 series cells, and the like. After obtaining the host cell transfected or transformed with the recombinant vector as described above, the antibody can be expressed by culturing under appropriate conditions, and then isolated to obtain the purified antibody.
In a preferred embodiment, the recombinant vector is the mammalian expression vector pBR322 and the host cell is a human kidney epithelial cell (293F cell).
In another embodiment of the invention, a human C-reactive protein antibody pair is provided, consisting of a first antibody and a second antibody as described above.
The first antibody and the second antibody provided by the invention are combined with different antigenic determinants on the surface of human CRP, are used for developing a double-antibody sandwich ELISA detection system, have the advantages of high specificity, high detection sensitivity and the like, can be used for measuring low CRP content and high CRP content, and have wide application range.
In a further embodiment, the invention provides the use of a human C-reactive protein monoclonal antibody or antibody pair as described above in the preparation of a human C-reactive protein detection reagent or kit.
The advantages of the human C-reactive protein monoclonal antibody or the antibody in the preparation of the human C-reactive protein detection reagent or the kit are the same as those of the human CRP monoclonal antibody described above in comparison with the prior art, and are not described in detail herein.
Based on the same inventive concept, the embodiment of the invention also provides a human C-reactive protein detection reagent or kit, which comprises the first antibody and/or the second antibody.
It should be emphasized that the first antibody and the second antibody may be used individually, or may be used together, or may be used in pairs. In the detection, for example, when the first antibody and/or the second antibody are used separately or in combination, the first antibody and/or the second antibody are used as the primary antibody or the capture antibody, and the sample to be detected is contacted with the first antibody and/or the second antibody, followed by detection of the antibody. In some embodiments, the first antibody and/or the second antibody may be conjugated to a detectable label, and qualitative or quantitative detection of CRP is achieved by analyzing the change in the identifiable signal produced by the detectable label. In other embodiments, the primary and/or secondary antibodies (as primary or capture antibodies) to human CRP are not labeled, and the detectable label is conjugated to a secondary antibody (as a detection antibody) or other molecule that can bind to the primary antibody, e.g., if the anti-human CRP antibody is a rabbit IgG antibody, the secondary antibody can be an anti-rabbit IgG antibody, thereby producing a change in the recognizable signal by the conjugation of the detectably labeled secondary antibody. When the antibodies are paired, one of the first antibody and the second antibody is used as a primary antibody or a capture antibody, and the other is used as a secondary antibody or a detection antibody.
The detection methods described above employ conventional immunological methods including, but not limited to: enzyme Immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot, immunohistochemical (IHC), immunofluorescence (IF), immunoblotting (WB), flow Cytometry (FC), and the like. The test subjects include recombinantly expressed CRP protein, cell secreted CRP protein, or CRP protein in human serum. The test sample includes, but is not limited to, CRP protein in serum, plasma, urine, cell culture fluid, and the like.
Preferably, the detection reagent or kit comprises a first antibody as a capture antibody (or primary antibody) and a second antibody as a detection antibody (or secondary antibody), and the second antibody is modified with a detectable label.
In particular, detectable labels for producing identifiable signal changes include, but are not limited to: biotin, fluorescein, chemiluminescent groups, fluorescent proteins (e.g., isophthalocyanin, B-phycoerythrin, perCP, and R-phycocyanin), enzymes (e.g., alkaline phosphatase, glucose oxidase, horseradish peroxidase, acid phosphatase), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies, or combinations thereof.
In a preferred embodiment, the detectable label is biotin.
The invention will be further illustrated with reference to specific examples. The experimental methods in which specific conditions are not specified in the following examples are generally conducted under conventional conditions, for example, those described in the molecular cloning Experimental guidelines (fourth edition) published in Cold spring harbor laboratory, or are generally conducted under conditions recommended by the manufacturer.
EXAMPLE 1 preparation of human CRP Rabbit monoclonal antibody
The immunogens used to prepare the Human CRP rabbit monoclonal antibodies of the present invention are derived from commercial recombinant Human CRP proteins (from Abclonal, accession number RP 01019) whose protein sequences are described in NCBI accession numbers: np_ 000558.2). The preparation method of the antibody is a monoclonal antibody development technology based on single B lymphocyte screening and culture, briefly, B lymphocytes are firstly sorted and cultured from immune antigen rabbit spleen cells, RNA in the B lymphocytes is extracted, the RNA is reversely transcribed into cDNA, natural paired heavy chain variable region (VH) genes and light chain variable region (VL) genes of the coding rabbit monoclonal antibodies are obtained through PCR amplification, finally the genes are respectively loaded on expression vectors, the vectors are co-transformed or transfected into host cells, the host cells are cultured, and the high-specificity and high-affinity rabbit antibodies A and B combined with human CRP are obtained through separation and purification.
The Amino Acids (AA) of antibody A and antibody B are shown in tables 1-2, respectively. For convenience of description, light chain CDR1-3 are denoted by LCDR1, LCDR2 and LCDR3, respectively, and heavy chain CDR1-3 are denoted by HCDR1, HCDR2 and HCDR3, respectively.
TABLE 1 sequence information for Rabbit monoclonal antibody A of this example
TABLE 2 sequence information of rabbit monoclonal antibody B of this example
The preparation method of the rabbit monoclonal antibody A and the rabbit monoclonal antibody B specifically comprises the following steps:
1. Animal immunization: 2 New Zealand white rabbits are immunized by taking recombinant Human CRP protein as an immunogen; each white rabbit is immunized by 200 mug, the immunogen is mixed with the equivalent complete Freund adjuvant to prepare an emulsifying agent before the first immunization, and the emulsifying agent is subcutaneously injected into the abdomen and the back of the rabbit at multiple points; 100 mug of immunogen is mixed with the equivalent amount of incomplete Freund's adjuvant every 3 weeks after the primary immunization to prepare an emulsifier, and the emulsifier is subcutaneously injected into the abdomen and the back of a rabbit for two times of boosting; serum samples of rabbits were collected after three immunizations, titers against human CRP were determined by ELISA, rabbits with high serum titers were boosted once by subcutaneous multipoint injection with 200 μg immunogen, animals were sacrificed three days later and spleens were taken.
2. Isolation of spleen cells: taking out a culture dish in a safety cabinet in a sterile operation manner, adding 30-40mL of basic culture medium, placing a cell screen, taking out spleen, placing the spleen in the cell screen, shearing superfluous connective tissue and fat on rabbit spleen tissue, cutting spleen tissue, placing the spleen tissue into the cell screen for grinding, taking a clean grinding rod, grinding the tissue with the tail end of the pressing part of the grinding rod to grind cells in a membrane slowly and freely, and suspending the tissue in a culture dish solution after the cell screen; the washed cell screen was washed with 10mL of basal medium and the basal medium outside the cell screen was collected. Centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 13mL of RBC erythrocyte lysate (purchased from BioGems company) at room temperature, gently blowing off cell clusters by using a pipettor, counting for 1min, performing erythrocyte lysis, adding 37mL of basic culture medium, uniformly mixing, stopping erythrocyte lysis, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 40mL of basic culture medium placed at room temperature, gently blowing off cell clusters by using a pipettor, resuspension cells, completing the first cleaning, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 20mL of basic culture medium placed at room temperature, gently blowing off cell clusters by using a pipettor, and resuspension cells; the resuspended cells were filtered again through a cell screen to remove agglomerated cells, after which the cells were counted.
3. B lymphocytes in spleen were isolated and B lymphocyte sorting was performed: the B lymphocytes in the spleen are isolated by adopting a conventional method, and related methods are disclosed in the patent (publication number: CN110016462A, publication date: 2019-07-16) and the patent (publication number: CN111518765A, publication date: 2020-08-11) of a B lymphocyte in vitro culture system and application thereof.
4. Cloning of the genes encoding the rabbit monoclonal antibodies: positive clones were identified by ELISA of CRP antigen-coated culture B lymphocyte supernatants to obtain antigen-specific B lymphocytes. Positive clone cells were collected and lysed, and RNA was extracted according to Quick-RNA TM MicroPrep kit instructions (purchased from ZYMO, cat# R1051) and reverse transcribed into cDNA, wherein the reverse transcription system included: oligo (dT) 12-18primer (Life) 1 μ L, dNTPs (10 mM) 1 μ L, RNA 11 μL; after 65℃for 5min in a PCR apparatus, 5 XFS Buffer (from ABconal) 4. Mu. L, DTT (100 mM) 1. Mu.L and RNase OUT (40U/. Mu.L) 1. Mu. L, ABScript II RT (200U/. Mu.L from ABconal) 1. Mu.L were added to the above products, and the mixture was mixed and reacted at 42℃for 1h,85℃for 5min to obtain cDNA. The cDNA is used as a template, a natural paired rabbit monoclonal antibody light chain variable region (VL) and heavy chain variable region (VH) are amplified from the cDNA of the corresponding positive clone by adopting a PCR method, and sequencing is carried out, and is completed by Jin Kairui biotechnology limited company. The PCR reaction system comprises: 4. Mu.L of cDNA, 1. Mu.L of forward primer (10 mM), 1. Mu.L of reverse primer (10 mM), 12.5. Mu.L of 2X Gloria HiFi (from ABclonal, cat. RK 20717) and 6.5. Mu. L H 2 O; the PCR amplification procedure included: the reaction mixture was subjected to preliminary denaturation at 98℃for 30s, followed by 40 cycles at 98℃for 10s,64℃for 30s, and 72℃for 30s, and finally kept at 72℃for 5min, and the resulting reaction mixture was kept at 4 ℃. Wherein primer sequences (5 '-3') for amplifying the VL and VH genes are shown below, and F and R represent forward primer and reverse primer, respectively:
VL-F: TGAATTCGAGCTCGGTACCCATGGACACGAGGGCCCCCAC (see SEQ ID NO. 21);
VL-R: CACACACACGATGGTGACTGTTCCAGTTGCCACCTGATCAG (see SEQ ID NO. 22);
VH-F: TGAATTCGAGCTCGGTACCCATGGAGACTGGGCTGCGCTG (see SEQ ID NO. 23);
VH-R: GTAGCCTTTGACCAGGCAGCCCAGGGTCACCGTGGAGCTG (see SEQ ID NO. 24).
5. Production and purification of rabbit monoclonal antibodies: in order to expand the rabbit monoclonal antibody for producing the human CRP protein, a mammal expression vector pBR322 loaded with genes carrying a light chain constant region (CL) and a heavy chain constant region (CH) is subjected to conventional linearization treatment by using XbaI and NheI restriction enzymes respectively, VL and VH genes which are amplified by PCR and contain signal peptides are purified, and then constructed into the expression vector by adopting a homologous recombination mode to obtain the light chain gene and heavy chain gene expression vector, and the construction of the expression vector is verified to be successful by sequencing. The expression patterns of the vectors used are shown in FIG. 1, pRB322 origin and f1 origin are replication promoters, AMPCILLIN is a resistance gene, CMVpromoter is a transcription promoter, SV40 PAterminator is a tailing signal, LIGHT CHAIN constant is the nucleic acid sequence of the light chain constant region (left panel), HEAVY CHAIN constant is the nucleic acid sequence of the heavy chain constant region (right panel). CL, CH genes CL is obtained by searching IMGT online database (www.imgt.org), searching rabbit source IGG GAMMA C REIGN for CH, searching rabbit source IGG KAPPA C REIGN.
The signal peptide of this example may be expressed by using an antibody commonly used in the art, such as a rabbit monoclonal antibody against Human interferon alpha 2 and its use (publication No. CN116063487A, publication No. 2023-05-05) and a light chain variable region upstream of a signal peptide "MDTRAPTQLLGLLLLWLPGATF" or "MDTRAPTQLLGLLLLWLPGARC" and a heavy chain variable region upstream of a signal peptide "METGLRWLLLVAVLKGVQC" of a high affinity Human IL-5 rabbit monoclonal antibody and its use (publication No. CN115819578A, publication No. 2023-03-21).
And (3) transfecting the expression vector which is verified to be correct by sequencing and contains the light chain gene and the heavy chain gene into 293F cells, and culturing for 72-96 hours after transfection to obtain the recombinant rabbit monoclonal antibody which contains the human CRP in the culture supernatant. The antibody of interest was purified from the culture supernatant using a proteona affinity gel resin (purchased from heaven and earth, cat No. SA 023100). Antibody purity > 95% was verified by electrophoresis on a 12% SDS-PAGE gel with an antibody A concentration of 1.92mg/mL and an antibody B concentration of 2mg/mL. The purified antibody is packaged and stored at a low temperature of-20 ℃ for standby.
Example 2 affinity test of Rabbit monoclonal antibodies A and B
After a plurality of Human CRP rabbit monoclonal antibodies are obtained, carrying out affinity test and epitope identification on the antibodies to obtain the antibodies A and B. The method comprises the following steps: the affinity of monoclonal antibodies A and B to human CRP protein was precisely determined using a Probe Life company Gator biomolecular interaction analyzer, with the concentration of antibodies A and B being 3 μg/mL. Antibodies were immobilized on protein a probes, respectively, and then binding was removed with recombinant Human CRP protein at two concentrations of 100nM and 200nM for antibody a and antibody B, respectively, to obtain affinity curves.
The affinity curves of antibodies a and B binding to human CRP are shown in fig. 2-3, respectively, wherein the ordinate represents the change in the thickness of the conjugate after the probe binds to the antibody and protein, and the abscissa represents the binding time, the dark gray curve is a real-time binding numerical curve, and the light gray curve is a fitted average curve. Affinity constants calculated by curve fitting are shown in table 3, dissociation coefficients K off are constants for characterizing the dissociation rates of antibodies and antigens, binding coefficients K on are constants for characterizing the binding rates of antibodies and their targets, and affinity constants K D are ratios of K off/Kon, representing equilibrium dissociation constants between antibodies and their antigens.
TABLE 3 determination of affinity-related parameters for Rabbit monoclonal antibodies
| Antibodies to | Kon(1/Ms) | KonError | Koff(1/s) | KoffError | KD(M) |
| A | 1.77×104 | 3.91×101 | 2.63×10-5 | 1.30×10-6 | 1.49×10-9 |
| B | 2.87×105 | 5.20×102 | 8.25×10-5 | 8.00×10-7 | 2.88×10-10 |
From the above results, the affinity constants K D of antibodies A and B with human CRP protein were 1.49X10 -9 M and 2.88X10 -10 M, respectively, and the affinity of the two antibodies was high.
Example 3 double antibody sandwich ELISA method and sensitivity test based on antibodies A and B
The method for establishing the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) by taking the antibody A as a capture antibody and the antibody B as a detection antibody comprises the following steps: 1) Coating the capture antibody A: diluting the antibody A to 0.5 mug/mL by using 1 XPBS, uniformly mixing by using a vortex instrument, adding into a 96-well micro-pore plate at 100 mug/well, covering a cover plate film, and placing in a refrigerator at 4 ℃ for incubation for 16-20h; 2) Washing the plate: after the incubation is completed, the liquid in the holes is discarded, the plate is washed once by 1 XPBST, 300 mu L of sample is added, the liquid in the holes is discarded after standing for 40s, and the liquid in the holes is dried on the flat paper; 3) Closing: adding E013 blocking solution (containing 2% BSA, 5% sucrose, 0.05% Tween and 0.1%proclin 300,pH7.2% in 1 XPBS) into the plate holes at a ratio of 200 μl/hole, covering with cover plate film, blocking at 37deg.C for 2h, discarding blocking solution, drying the ELISA plate, oven drying at 37deg.C for 0.5-2h, and taking out; 4) Adding antigen protein: human CRP protein was gradient diluted with E013 blocking solution, dilution concentration: 2000. 1000, 500, 250, 125, 62.5, 31.25 and 0pg/mL, then sequentially adding different concentrations into the ELISA plate at 100 mu L/well, covering a cover plate film, and incubating at 37 ℃ for 2 hours; 5) Washing the plate: after the incubation is completed, the liquid in the holes is discarded, the plate is washed three times by 1 XPBST, 300 mu L of sample is added, the liquid in the holes is discarded after standing for 40s, and the liquid in the holes is dried on the flat paper; 6) Adding detection antibody B: diluting antibody B (B-biotin) marked by biotin into 0.0556 mu g/mL, sequentially adding 100 mu L/hole into an ELISA plate, covering a cover plate film, and incubating at 37 ℃ for 1h; The B-biotin processing method comprises the following steps: preparing an antibody B into a solution with the concentration of 1mg/mL, and preparing NHS-LC-biotin into a solution with the concentration of 60mg/mL by using DMSO; 200 mu L of 1mg/mL of antibody B solution is taken, and 10 mu L of 60mg/mL of NHS-LC-biotin solution is added; after mixing, the mixture was left at room temperature for 30min, and then 50. Mu.g of 500mM Tris solution (pH 9.0) was added to stop the reaction; finally adding a large amount of 1 XPBS buffer solution with pH of 7.4, centrifuging by using a centrifugal column with the exclusion limit of 30KD, and removing redundant biotin molecules and balancing a buffer solution system; 7) Washing the plate: after the incubation is completed, the liquid in the holes is discarded, the plate is washed three times by 1 XPBST, 300 mu L of sample is added, the liquid in the holes is discarded after standing for 40s, and the liquid in the holes is dried on the flat paper; 8) Adding SA-HRP: 100 xSA-HRP (horseradish peroxidase labeled streptavidin, available from Wuhan Sanying biotechnology Co., ltd., product No. SA 00001-0) concentrate is diluted 100 times, and added into an ELISA plate sequentially at 100 μl/hole, covered with a cover plate film, and incubated at 37deg.C for 0.5 hr; 9) Washing the plate: after the incubation is completed, the liquid in the holes is discarded, the plate is washed three times by 1 XPBST, 300 mu L of sample is added, the liquid in the holes is discarded after standing for 40s, and the liquid in the holes is dried on the flat paper; 10 Adding TMB color development liquid: adding 3,3', 5' -tetramethyl benzidine (TMB) color development liquid into an ELISA plate at a concentration of 100 mu L/hole, covering a cover plate film, and incubating at 37 ℃ for 15min;11 Reading: after the incubation was completed, the microplate was removed, 50. Mu.L of stop solution (1 mol/L hydrochloric acid) was added to each well, and immediately reading was performed with an microplate reader. The calibration value Y1 (y1=od 450nm-OD630nm) of the absorbance was plotted on the abscissa with the human CRP protein concentration, and a standard curve was obtained, see fig. 4. The lowest human CRP concentration with an average absorbance greater than the average absorbance of the triplicate blank control was the sensitivity of the double antibody sandwich ELISA and the results are shown in table 4.
TABLE 4 sensitivity test data for the establishment of double antibody sandwich ELISA based on rabbit monoclonal antibodies A and B
The results show that the sensitivity (sensitivity) of CRP protein detection by establishing double-antibody sandwich ELISA based on the antibodies A and B reaches 25.97pg/mL, the linear range is 31.2-2000pg/mL, and R 2 = 0.9928, so that the high-sensitivity detection of trace protein can be satisfied.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A human C-reactive protein monoclonal antibody, wherein the monoclonal antibody is a first antibody or a second antibody, and wherein:
The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the first antibody are respectively shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are respectively shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10;
The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the second antibody are shown as SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17 respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20 respectively.
2. The monoclonal antibody of claim 1, wherein the amino acid sequence of the first antibody light chain variable region is shown in SEQ ID No.3 and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 4;
The amino acid sequence of the light chain variable region of the second antibody is shown as SEQ ID NO.13, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 14.
3. The human C-reactive protein monoclonal antibody of claim 2, wherein the amino acid sequence of the first antibody light chain is shown in SEQ ID No.1 and the amino acid sequence of the heavy chain is shown in SEQ ID No. 2;
The amino acid sequence of the light chain of the second antibody is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 12.
4. The human C-reactive protein monoclonal antibody of claim 1, wherein the first antibody or the second antibody is a full-length antibody or an antibody fragment;
The antibody fragment is selected from at least one of Fab fragment, F (ab) 2 fragment, fv fragment, (Fv) 2 fragment, scFv fragment and sc (Fv) 2 fragment.
5. A nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the first antibody or the second antibody of any one of claims 1-4.
6. A human C-reactive protein antibody pair, consisting of a first antibody according to any one of claims 1-4 and a second antibody.
7. Use of a human C-reactive protein monoclonal antibody according to any one of claims 1-4 or a human C-reactive protein antibody according to claim 6 for the preparation of a human C-reactive protein detection reagent or kit.
8. A human C-reactive protein detection reagent or kit comprising the human C-reactive protein monoclonal antibody of any one of claims 1-4 or the human C-reactive protein antibody pair of claim 6.
9. The human C-reactive protein assay reagent or kit of claim 8, wherein the assay reagent or kit comprises a first antibody and a second antibody, the first antibody is a capture antibody, the second antibody is a detection antibody, and the second antibody is modified with a detectable label.
10. The human C-reactive protein assay reagent or kit of claim 9, wherein the detectable label is biotin.
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