CN117229395A

CN117229395A - Antibody specifically binding to calcitonin and application thereof

Info

Publication number: CN117229395A
Application number: CN202111074126.XA
Authority: CN
Inventors: 徐伟玲; 陈自敏; 李丽华; 杨晴; 梁梅芳; 宋浏伟; 熊君辉; 孙旭东
Original assignee: Xiamen Yingbomai Biotechnology Co ltd; Xiamen Innodx Biotech Co ltd
Current assignee: Xiamen Yingbomai Biotechnology Co ltd; Xiamen Innodx Biotech Co ltd
Priority date: 2021-09-14
Filing date: 2021-09-14
Publication date: 2023-12-15

Abstract

The present invention relates to monoclonal antibodies, conjugates and kits that specifically bind calcitonin. The invention also relates to a detection method of the calcitonin antigen, in particular to a detection method by a chemiluminescence method. The monoclonal antibodies of the invention are useful for the detection of calcitonin and for the diagnosis of diseases caused by changes in calcitonin levels.

Description

Antibody specifically binding to calcitonin and application thereof

Technical Field

The present invention relates to the fields of immunology and molecular virology, in particular to the detection of calcitonin. In particular, the invention relates to monoclonal antibodies, conjugates and kits that specifically bind to calcitonin. The invention also relates to a detection method of the calcitonin antigen, in particular to a detection method by a chemiluminescence method. The invention also relates to the diagnosis of diseases caused by changes in calcitonin levels.

Background

Calcitonin (CT) is produced and secreted by the parathyroid follicular cells (bright cells or C cells) and consists of 32 amino acids with a disulfide bond between cysteines at positions 1,7, and has a molecular weight of about 3418, which is secreted primarily by the parathyroid follicular cells (C cells). It is metabolized in the liver and kidney, regulated by serum calcium levels, and has the main functions of inhibiting bone resorption by osteoclasts, reducing the level of blood calcium, and preventing bone loss during calcium stress (e.g., pregnancy, lactation and growth). The serum calcitonin concentration of normal people is 10-20pg/ml.

The determination of the calcitonin content is of great significance for the evaluation of osteoporosis and the detection of thyroid function. Elevated levels of calcitonin can be seen in a variety of pathological conditions, the most significant clinical condition associated with disordered mass secretion of calcitonin being Medullary Thyroid Carcinoma (MTC), which is a low incidence but high mortality, possibly sporadic, and possibly autosomal dominant inherited disease. By detecting the serum calcitonin level, the MTC can be diagnosed early, so that complete cure of early subclinical diseases is possible, and the serum calcitonin level is also an important index for observing and evaluating the curative effect of treatment and prognosis. Abnormal elevation of calcitonin levels can also be seen in lung cancer, particularly small cell lung cancer, breast cancer, tumors of neural kurtosis origin, hyperthyroidism, hypergastrinemia, renal failure and chronic inflammatory diseases.

Calcitonin (CT) and Procalcitonin (PCT) have the same amino acid sequence at positions 60-91, but are expressed in different forms in human bodies, and have different structures, so that functions are different. However, due to the same amino acid sequence, we used specific monoclonal antibodies to screen as capture monoclonal antibodies to avoid PCT cross infection, and simultaneously screened CT, PCT cross-reactive high affinity epitope monoclonal antibodies as label monoclonal antibodies to form chemiluminescent reagents of the double antibody sandwich method. Since the main amino acid sequence of CT is 60-91 of the amino acid sequence of PCT, detection of CT requires screening specific antibodies to avoid cross-interference of PCT. The current detection method of calcitonin adopts a specific calcitonin antibody to capture and adopts a double antibody sandwich method formed by using an epitope antibody crossed by CT and PCT as a label, and the method ensures the detection specificity.

Thus, there is a need in the art to develop specific antibodies against human calcitonin.

Disclosure of Invention

The inventors of the present application have made a great deal of experiments and repeated experiments to obtain an antibody capable of specifically binding to calcitonin. Based on this, the inventors have made a great deal of creative work to pair-detect these antibodies, and developed a detection kit capable of detecting calcitonin with high sensitivity. Therefore, the antibody of the application has the application of detecting the calcitonin, can be used for detecting the potential of diseases caused by the change of the calcitonin level, and has great clinical value.

In a first aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to Calcitonin (CT), comprising:

(a) A heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs):

(i) VH CDR1 consisting of the sequence: SEQ ID NO. 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto,

(ii) VH CDR2 consisting of the sequence: SEQ ID NO. 2, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared to it, and

(iii) VH CDR3 consisting of the sequence: SEQ ID NO. 3, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto;

and/or the number of the groups of groups,

(b) A light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs):

(iv) VL CDR1, consisting of the sequence: SEQ ID NO. 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto,

(v) VL CDR2, consisting of the sequence: SEQ ID NO. 5, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto, and

(vi) VL CDR3 consisting of the sequence: SEQ ID NO. 6, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto.

In certain embodiments, the substitutions described in any one of (i) - (vi) are conservative substitutions.

In certain embodiments, an antibody, or antigen-binding fragment thereof, as described previously, comprises:

(a) A heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i) SEQ ID NO: 7;

(ii) And SEQ ID NO:7 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or (b)

(iii) And SEQ ID NO:7, a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and

(b) A light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO: 8;

(v) And SEQ ID NO:8 (e.g., substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) as compared to a sequence having one or more amino acid substitutions, deletions, or additions; or (b)

(vi) And SEQ ID NO:8, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

In certain embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

In certain embodiments, the antibody or antigen binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO. 7 and a VL comprising the sequence shown as SEQ ID NO. 8.

In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to Calcitonin (CT), comprising:

(i) VH CDR1 consisting of the sequence: SEQ ID NO. 10, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto,

(ii) VH CDR2 consisting of the sequence: 11, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and

(iii) VH CDR3 consisting of the sequence: 12, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto;

And/or the number of the groups of groups,

(iv) VL CDR1, consisting of the sequence: SEQ ID NO. 13, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto,

(v) VL CDR2, consisting of the sequence: SEQ ID NO. 14, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto, and

(vi) VL CDR3 consisting of the sequence: SEQ ID NO. 15, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto.

(i) SEQ ID NO:16, a sequence shown in seq id no;

(ii) And SEQ ID NO:16 (e.g., substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) as compared to a sequence having one or more amino acid substitutions, deletions, or additions; or (b)

(iii) And SEQ ID NO:16, a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

and

(iv) SEQ ID NO:17, a sequence shown in seq id no;

(v) And SEQ ID NO:17 (e.g., substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) as compared to a sequence having one or more amino acid substitutions, deletions, or additions; or (b)

(vi) And SEQ ID NO:17, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

In certain embodiments, the antibody or antigen binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO. 16 and a VL comprising the sequence shown as SEQ ID NO. 17.

In certain embodiments, an antibody or antigen binding fragment thereof as described previously, is murine or humanized.

In certain embodiments, the antibody or antigen binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin.

In certain embodiments, the antibody or antigen binding fragment thereof comprises a constant region derived from a human immunoglobulin.

In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., igG1, igG2, igG3, or IgG 4), and/or the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (e.g., kappa or lambda).

In another aspect, the application provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof, as described above.

In another aspect, the application provides a vector comprising a nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

In another aspect, the application provides a host cell comprising a nucleic acid molecule as described above or a vector as described above.

In another aspect, the application provides a method of producing an antibody or antigen-binding fragment thereof as described above, comprising culturing a host cell as described above under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

In another aspect, the application provides a conjugate comprising an antibody or antigen-binding fragment thereof as described above, and a detectable label attached to or coated with the antibody or antigen-binding fragment thereof.

In certain embodiments, the detectable label is selected from the group consisting of an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridine esters, luminol and derivatives thereof, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, biotin, or a magnetic bead (e.g., an immunomagnetic bead with an amino group, an immunomagnetic bead with a carboxyl group, an immunomagnetic bead with a hydroxyl group, an immunomagnetic bead with an aldehyde group).

In certain embodiments, the detectable label is a magnetic bead.

In certain embodiments, the detectable label is an acridine ester compound.

In another aspect, the application provides a kit comprising: (i) a first antibody or first conjugate, or (ii) a second antibody or second conjugate, or (iii) a combination thereof.

In certain embodiments, the first antibody is an antibody or antigen-binding fragment thereof as described above, and the first conjugate is a conjugate as described above;

The second antibody is an antibody capable of binding to a different epitope of Calcitonin (CT); the second conjugate includes a second antibody and a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound, luminol and derivatives thereof, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin. In certain embodiments, the second conjugate comprises a second antibody and an acridinium ester.

In certain embodiments, the second antibody is capable of binding amino acids 16, 17, 19 and/or 20 of Calcitonin (CT).

In certain embodiments, the second antibody is an antibody or antigen-binding fragment thereof as described above.

In certain embodiments, the second conjugate is a conjugate as described previously.

In another aspect, the application provides a method for detecting the presence or level of calcitonin in a biological sample, comprising:

which comprises the use of an antibody or antigen binding fragment thereof as described above or a conjugate as described above or a kit as described above.

In certain embodiments, the method is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

In certain embodiments, the method comprises: contacting the sample with a first antibody or conjugate thereof that binds a different epitope of the calcitonin antigen, and a second antibody or conjugate thereof, and qualitatively or quantitatively detecting binding of the two antibodies to the calcitonin antigen, wherein binding is indicative of the presence or concentration of the calcitonin antigen in the sample.

In certain embodiments, the first antibody is an antibody or antigen-binding fragment thereof as described above.

In certain embodiments, the first conjugate is a conjugate as described previously.

In another aspect, the application provides the use of an antibody or antigen-binding fragment thereof as described above in the preparation of a kit for detecting the presence or level of calcitonin in a sample and/or for diagnosing whether a subject has a disease caused by altered calcitonin levels (e.g. osteoporosis, medullary thyroid cancer, lung cancer, small cell lung cancer, breast cancer, tumors of neuronal origin, hyperthyroidism, hypergastrinemia, renal failure and chronic inflammatory disease).

In certain embodiments, the kit is a kit as described previously.

In certain embodiments, the kit detects the presence or level of calcitonin in a sample by a method as described previously.

In certain embodiments, the sample is a blood sample (e.g., whole blood, plasma, or serum), fecal matter, oral or nasal secretions, or alveolar lavage from a subject (e.g., a mammal, preferably a human).

In certain embodiments, the calcitonin has a sequence as shown in SEQ ID NO. 9.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Further, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA, etc., as used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "Calcitonin (CT)" refers to a polypeptide hormone secreted by thyroid parafollicular cells (C cells). Calcitonin is a specific and sensitive tumor marker for diagnosing and monitoring medullary thyroid cancer. The sequences of which can be obtained from a common database.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each V is _H And V _L By the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites, respectively. Amino acidsThe assignment of the regions or domains can be carried out by Kabat, sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987 and 1991)), or Chothia&Lesk (1987) J.mol.biol.196:901-917; chothia et al (1989) Nature 342:878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs, designated CDR1, CDR2 and CDR3, are contained in each of the variable regions of the heavy and light chains. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, e.g., as in the Kabat numbering system (Kabat et al, sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md, 1991), the Chothia numbering system (Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al, dev. Comparat. Immunol.27:55-77,2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, for example, lefranc et al, dev. Comparat. Immunol.27:55-77,2003).

In the present invention, the CDRs contained in the antibodies or antigen binding fragments thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by an antibody or antigen binding fragment thereof of the invention are preferably determined by Kabat, chothia or IMGT numbering system. In certain embodiments, the CDRs contained in an antibody or antigen binding fragment thereof of the invention are preferably determined by the Kabat numbering system.

As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion. See generally Fundamental Immunology, ch.7 (Paul, W., ed., 2 nd edition, raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes, antigen binding fragments of antibodies may be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies non-limiting examples of antigen binding fragments include Fab, fab ', F (ab') ₂ Fd, fv, complementarity Determining Region (CDR) fragments, scFv, diabodies (diabodies), single domain antibodies (single domain antibody), chimeric antibodies, linear antibodies (linear antibodies), nanobodies (technology from Dommantis), probody and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptide. Engineered antibody variants are reviewed in Holliger et al, 2005; nat Biotechnol, 23:1126-1136.

As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al Nature 341:544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') ₂ Fragment "means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab 'fragment" means a reduction-linked F (ab') ₂ The resulting fragment after disulfide bonding of the two heavy chain fragments in the fragment consists of one complete light and heavy chain Fd fragment (consisting of VH and CH1 domains).

As used herein, the term "Fv" means an antibody fragment consisting of VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragment that forms the complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even one variable region (e.g., fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the complete binding site.

As used herein, the term "Fc" means an antibody fragment formed by disulfide bonding of the second and third constant regions of a first heavy chain of an antibody with the second and third constant regions of a second heavy chain. The Fc fragment of an antibody has a number of different functions, but does not participate in antigen binding.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH domains are linked by a linker (linker) (see, e.g., bird et al, science 242:423-426 (1988); huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckaphun, the Pharmacology of Monoclonal Antibodies, volume 113, roseburg and Moore, springer-Verlag, new York, pages 269-315 (1994)). Such scFv molecules may have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a polypeptide having an amino acid sequence (GGGGS) can be used ₄ Variants thereof may be used (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J.Immunol.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56 and Roovers et al (2001), cancer Immunol. In some cases, disulfide bonds may also exist between VH and VL of scFv. In certain embodiments of the invention, an scFv may form a di-scFv, which refers to two or more individual scFv in tandem to form an antibody. In certain embodiments of the invention, scFv may be formed (scFv) ₂ It refers to the formation of antibodies from two or more individual scfvs in parallel.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. The percent identity between two amino acid sequences can also be determined using the algorithms of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which have been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package (available on www.gcg.com), using the Blossum 62 matrix or PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al Protein Eng.12 (10): 879-884 (1999); and Burks et al Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

The twenty conventional amino acids referred to herein are written following conventional usage. See, e.g., immunology-a Synthesis (2nd Edition,E.S.Golub and D.R.Gren,Eds, sinauer Associates, sundland, mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "subject" refers to a mammal, such as a human. In certain embodiments, the subject (e.g., human) has, or is at risk of having, a SARS-CoV-2 infection or a disease associated with a SARS-CoV-2 infection (e.g., COVID-19).

As used herein, the term "epitope" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "epitopes" are also known in the art as "antigenic determinants". Epitopes or antigenic determinants are generally composed of chemically active surface groupings of molecules such as amino acids or carbohydrates or sugar side chains, and generally have specific three dimensional structural characteristics as well as specific charge characteristics. For example, an epitope typically comprises at least 3,4,5,6,7,8,9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which may be "linear" or "conformational. See, e.g., epitope Mapping Protocols in Methods in Molecular Biology, volume 66, g.e.Morris, ed. (1996). In linear epitopes, the points of all interactions between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In conformational epitopes, points of interaction exist across amino acid residues of the protein that are separated from each other.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. In certain embodiments, an antibody that specifically binds to (or has specificity for) an antigen means that the antibody binds to or has specificity for an antigen in an amount of less than about 10 ^-5 M, or less, binds the antigen with an affinity (KD).

Advantageous effects of the application

The present application provides monoclonal antibodies capable of specifically binding calcitonin. The monoclonal antibodies can be used to detect the presence or level of calcitonin and can be used to diagnose diseases caused by changes in calcitonin levels (e.g., osteoporosis, medullary thyroid cancer). The kit prepared based on the monoclonal antibody of the application can detect the existence or the level of the calcitonin in a sample with high sensitivity, and can detect the calcitonin as low as or lower than 0.5 pg/ml.

Embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present application and are not to be construed as limiting the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

FIG. 1 shows the detection results of the chemiluminescent kit and Roche reagent detection WHO standard for antibody preparation of the present application.

Sequence information

The information of the partial sequences to which the present application relates is provided in table 1 below.

Table 1: description of the sequence

Detailed Description

The application will now be described with reference to the following examples, which are intended to illustrate the application, but not to limit it.

The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. For example, for the conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present application, reference may be made to Sambrook (Sambrook), friech (Fritsch) and manitis (Maniatis), molecular cloning: laboratory Manual (MOLECULAR CLONING: A LABORATORY MANUAL), edit 2 (1989); the handbook of contemporary molecular biology (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (edited by f.m. ausubel (f.m. ausubel) et al, (1987)); series (academic publishing company) of methods in enzymology (METHODS IN ENZYMOLOGY): PCR 2: practical methods (PCR 2:A PRACTICAL APPROACH) (M.J. MaxFrson (M.J. MacPherson), B.D. Hemsl (B.D. Hames) and G.R. Taylor (G.R. Taylor) editions (1995)), and animal cell CULTURE (ANIMAL CELL CULTURE) (R.I. French Lei Xieni (R.I. Freshney) editions (1987)).

In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

EXAMPLE 1 preparation of CT antigen

1.1 Synthesis of CT antigen (the amino acid sequence of which is shown in SEQ ID NO: 9), wherein CT is a protein having 32 amino acids, BSA was coupled to CT antigen to form BSA-CT (obtained from Shanghai Co., ltd. In biological engineering) in order to enhance its immunity.

EXAMPLE 2 preparation of anti-Callitonin mouse monoclonal antibody

2.1 immunization of mice

BSA-CT antigen was diluted to 0.4mg/mL and mixed with Freund's adjuvant in equal volume to form a water-in-oil emulsion (method for judging whether the mixture was completely emulsified, a small drop of the mixture was allowed to be dropped on the surface of clear water, and if the mixture was not coagulated, it was considered to be substantially uniformly mixed). The primary immunization used Freund's complete adjuvant, the subsequent booster immunization used Freund's incomplete adjuvant, and the last booster immunization 72h prior to fusion was unadjuvanted.

Basic immunization of mice: the immunogens described above were used to immunize 6-8 week old BALB/c females by subcutaneous multiple injections at a dose of 500. Mu.L/dose per time, and 200. Mu.L of venous blood was collected prior to each immunization for titer determination. Immunization was boosted every 2 weeks. Serum titers were determined by indirect ELISA, and when the mice reached the plateau, the mice stopped immunization and were ready to develop fusion.

2.2 preparation and screening of fusion hybridomas

After 72h boosting before fusion, the spleen of the mouse is taken to prepare a cell suspension and is subjected to cell fusion with the Sp2/0 of the myeloma cells of the mouse so as to obtain hybridoma cells. Feeder cells were prepared prior to this. In the culture process of hybridoma cells, a great number of myeloma cells and spleen cells after fusion die in 1640-HAT culture solution, single cells or a small number of scattered cells are not easy to survive, other cells must be added to enable the cells to survive, and the added living cells are called feeder cells. The laboratory used mouse peritoneal macrophages or 13 day old mouse thymocytes as feeder cells.

2.2.1 preparation of mouse macrophages: the method comprises the following steps: (i) One BALB/c mouse with age of about 6 weeks is killed by introducing neck, washed by tap water and soaked In 75% ethanol solution for 5min; placing the mice in an ultra-clean workbench to enable the abdomen of the mice to face upwards; the abdomen skin of the mouse is lifted by forceps, a small opening is cut, and the skin is torn open in the up-down direction by large forceps, so that the abdomen is fully exposed. (ii) The peritoneum was lifted with sterile ophthalmic forceps, then a proper amount of culture solution was injected into the abdominal cavity with a 5mL syringe, the mouse limb was gently lifted with another sterile ophthalmic forceps, and finally the culture solution was aspirated into the centrifuge tube with the syringe. (iii) Dissolving abdominal cavity cell solution in HAT culture solution or HT culture solution to give concentration of 2×10 ⁵ Macrophaga feeder cells per mL. (iv) Adding 96-well cell culture plates, and culturing in an incubator with 0.1mL of each well; or directly mixed with the fused cells and added to a 96-well cell culture plate.

2.2.2 preparation of mouse thymocytes: the method comprises the following steps of (i) killing a BALB/c mouse with a neck of about 13 days old, flushing with tap water, and soaking in 75% ethanol solution for 5min; placing the mice in an ultra-clean workbench to enable the abdomen of the mice to face upwards; (ii) The skin of the abdomen of the mouse was lifted with forceps and the outer skin of the abdomen and chest was cut off. (iii) The chest was cut with another pair of clean scissors, the milky thymus was removed with forceps, and the mixture was ground and passed through a 200 mesh cell sieve to obtain thymus feeder cell sap.

2.2.3 preparation of mouse myeloma cells: generally, the recovery of myeloma cells of mice is started 5 days before fusion, and about 6 bottles of myeloma cells are required to be 35cm for each fusion ² Sp2/0 cells.

2.2.4 preparation of immune splenocytes: the method comprises the following steps of. (i) BALB/C mice to be fused are taken, the mice die due to eyeball bloodletting are removed, and the collected blood is made into antiserum which can be used as a positive control for antibody detection. Washing with tap water, soaking in 75% ethanol solution for 5min, and placing on the dissecting plate of mice in the super clean bench. (ii) The spleen was removed by opening the abdominal cavity in a sterile operation, cutting into small pieces with scissors, placing on a 200 mesh cell screen, squeezing and grinding with a grinding rod (plunger), and simultaneously dripping RPMI-1640 culture solution with a blowpipe. (iii) Adding an appropriate amount of RPMI-1640 culture solution, standing for 3-5min, taking 2/3 part of suspension, and transferring into a 50mL plastic centrifuge tube. The above process is repeated for 2-3 times. (iv) Cells were washed 3 times (1000 rpm. Times.10 min) with RPMI-1640 medium. (v) cells were resuspended in RPMI-1640 medium and counted.

2.2.5 preparation of hybridomas using PEG fusion promoter fusion: the method comprises the following steps of. (1) 1mL of PEG-1500 and 10mL of RPMI-1640 serum-free medium and 200mL of complete medium were pre-warmed to 37℃prior to fusion. (ii) The prepared myeloma cells and spleen cells were mixed in 50mL centrifuge tubes (1X 10) ⁸ Splenocyte +1×10 ⁷ Myeloma cells, about 10:1), centrifuged at 1500rpm x 8min, and after centrifugation, gently flicked the bottom of the tube to loosen the cells into a paste. (iii) A1 mL pipette aspirates 0.8mL (1X 10) ⁸ Splenocytes +0.8mL PEG) were added to the centrifuge tube with gentle agitation, and the PEG was added over 60 seconds on average, followed by 10mL RPMI-1640 complete broth pre-warmed to 37 ℃ with gentle agitation. Finally, RPMI-1640 culture solution was added to 40mL, and the mixture was centrifuged at 1000rpm X5 min. (iv) The supernatant was discarded, a small amount of HT medium was carefully removed from the cells, the cells were transferred to the prepared HT medium, 96 well cell culture plates were added, 0.1mL per well, and placed in a CO2 incubator for culturing. (v) After 12h, preparing a proper amount of HAT complete culture medium, and dropwise adding 0.1mL into each hole; after 5 days, 50-100% exchange of the cell supernatant in the wells with HT complete medium; after about 9-14 days, the supernatant was aspirated for detection.

2.2.6 selection of hybridomas: indirect ELISA screening, coating recombinant antigen 100ng/mL, coating 0.1mL for each well, adding cell supernatant 50uL for detection, and selecting positive cloning wells.

2.2.7 cloning of hybridoma cells: the limiting dilution method is adopted, the cells are subjected to gradient dilution according to a certain concentration, and then inoculated into each well of a 96-well cell culture plate, so that only one cell grows in the well as much as possible. Hybridoma monoclonal positive cell lines typically require repeated cloning 2-3 times until 100% positive is confirmed as stable clones.

2.2.8 mab screening: the cells were subjected to three stable cloning to obtain stable cell lines, cultured in large amounts, cryopreserved, induced mice to develop ascites, and purified by an affinity column to obtain 7 monoclonal antibodies with a purity of greater than 90%, which were designated as 13C2-2, 11A6-2, 10B11, 14D11, 13A6,3A2 and 8F6, respectively, and stored in 20mM PBS solution, respectively.

Acquisition of 2.2.9 antibody sequences

10B11 hybridoma cells cultured to the logarithmic growth phase were subjected to RNA extraction by Trizol method (Invitrogen), and the final extracted RNA pellet was dissolved in 50. Mu.l DEPC water. Then, reverse transcription PCR was performed on each of the heavy chain variable region and the light chain variable region, and the PCR product was recovered and sequenced. The sequences were blast aligned to determine the heavy and light chain variable region sequences of the antibodies. The amino acid sequences of the finally determined variable regions of 10B11 are shown in SEQ ID NOS.7 and 8 of Table 1. The amino acid sequences of the finally determined variable regions of 14D11 are shown in SEQ ID NOS.16 and 17 of Table 1.

Further, the CDR sequences of mouse monoclonal antibody 10B11 were also determined using the method described by Kabat et al (Kabat et al, sequences of Proteins of Immunological Interest, fifth edition, public Health Service, national institutes of health, beziida, maryland (1991), pages 647-669). The amino acid sequences of the CDRs of the finally determined variable region of 10B11 are shown in SEQ ID NOS: 1-6 of Table 1. The amino acid sequences of the CDRs of the finally determined variable region of 14D11 are shown in SEQ ID NOS: 10-15 of Table 1.

Example 3 in vitro epitope identification of anti-calcitonin mouse monoclonal antibody

3.1 polypeptide Synthesis and Ala epitope mutant Synthesis

3.1.1 26 polypeptides (from Shanghai Co., ltd. Of the engineering bioengineering) were synthesized using the CT sequence as a reference sequence. Together, these 26 polypeptides (CT 1-CT 26) cover the full length of 32 amino acids of Callitonin. The polypeptide information for CT1-CT32 is shown in Table 2 below. Based on the further results of the polypeptides, single alanine mutations were performed for amino acids CT9-24, CT24-32, and the mutated amino acid synthesis is shown in Table 3 below.

TABLE 2 epitope sequences of CT1-CT32

TABLE 3 mutant epitope sequences

Name of the name	Amino acid position	Amino acid sequence	SEQ ID NO:
				CT27	CT924-1	AGTYTQDFNKFHTFPQ	33
CT28	CT924-2	LATYTQDFNKFHTFPQ	34
				CT29	CT924-3	LGAYTQDFNKFHTFPQ	35
CT30	CT924-4	LGTATQDFNKFHTFPQ	36
				CT31	CT924-5	LGTYAQDFNKFHTFPQ	37
CT32	CT924-6	LGTYTADFNKFHTFPQ	38
				CT33	CT924-7	LGTYTQAFNKFHTFPQ	39
CT34	CT924-8	LGTYTQDANKFHTFPQ	40
				CT35	CT924-9	LGTYTQDFAKFHTFPQ	41
CT36	CT924-10	LGTYTQDFNAFHTFPQ	42
				CT37	CT924-11	LGTYTQDFNKAHTFPQ	43
CT38	CT924-12	LGTYTQDFNKFATFPQ	44
				CT39	CT924-13	LGTYTQDFNKFHAFPQ	45
CT40	CT924-14	LGTYTQDFNKFHTAPQ	46
				CT41	CT924-15	LGTYTQDFNKFHTFAQ	47
CT42	CT924-16	LGTYTQDFNKFHTFPA	48
				CT43	CT2432-1	ATAIGVGAP-NH2	49
CT44	CT2432-2	QAAIGVGAP-NH2	50
				CT45	CT2432-4	QTAAGVGAP-NH2	51
CT46	CT2432-5	QTAIAVGAP-NH2	52
				CT47	CT2432-6	QTAIGAGAP-NH2	53
CT48	CT2432-7	QTAIGVAAP-NH2	54
				CT49	CT2432-9	QTAIGVGAA-NH2	55

3.2 epitope validation and Ala epitope mutation competition result analysis of anti-Callitonin mouse monoclonal antibody

The 3.2.1CT antigen is diluted by carbonic acid buffer solution (20 mM CB, pH 9.6) and coated on a polyvinyl chloride plate, and the seven antibodies purified by the protein A column and the synthesized CT truncated antigen are mixed and incubated for 30min according to a certain proportion (the synthesized sequences are respectively supplemented at last) (50 ul of 100ng/ml antibody: 50ul of 50ug/ml truncated antigen) to obtain a detection sample; the control sample was incubated for 30min with a proportion of antibody to 20mM PBS (50 ul of 100ng/ml antibody: 50ul of 20mM PBS), the incubated test and control samples were then added to the CT antigen plate for 30min, the plate was washed 5 times, GAM-HRP (1/5000 dilution) was added for 30min, the plate was washed 5 times, and the substrate was added for 15min. And finally, calculating and analyzing the detected OD value by reading the value under the wavelength of 450/620 of the enzyme label instrument, wherein a calculation formula is (OD value of a control sample-OD value of a detection sample)/OD value of the control sample, and the judgment standard is as follows: more than 90% showed good competition, indicating that the antibodies should recognize the epitope, and the data analysis and evaluation are shown in tables 4 and 5 below.

TABLE 4 analysis of competitive results

Name of the name

Amino acid position

Amino acid sequence

8F6

13A6

14D11

3A2

CT1

aa1-aa16

CGNLSTCMLGTYTQDF

11％

30％

1％

14％

CT2

aa9-aa24aa

LGTYTQDFNKFHTFPQ

98％

99％

CT3

17-32aa

NKFHTFPQTAIGVGAP

5％

18％

32％

27％

CT4

aa12-aa23

YTQDFNKFHTFP

93％

98％

99％

100％

CT5

aa13-aa22

TQDFNKFHTF

-4％

86％

91％

93％

CT6

aa13-aa23

TQDFNKFHTFP

16％

97％

94％

95％

CT7

aa13-aa24

TQDFNKFHTFPQ

94％

96％

95％

CT8

aa14-aa22

QDFNKFHTF

-37％

93％

92％

91％

CT9

aa14-aa23

QDFNKFHTFP

11％

96％

95％

CT10

aa14-aa24

QDFNKFHTFPQ

93％

91％

95％

92％

CT11

aa15-aa20

DFNKFH

20％

78％

68％

78％

CT12

aa15-aa21

DFNKFHT

21％

93％

92％

CT13

aa15-aa22

DFNKFHTF

-18％

92％

CT14

aa15-aa23

DFNKFHTFP

-66％

95％

91％

90％

CT15

aa15-aa24

DFNKFHTFPQ

69％

93％

91％

96％

CT16

aa16-aa19

FNKF

17％

14％

11％

-8％

CT17

aa16-aa20

FNKFH

15％

-5％

-14％

-32％

CT18

aa16-aa21

FNKFHT

22％

6％

30％

-1％

TABLE 5 analysis of competitive results

Name of the name	Amino acid position	Amino acid sequence	13C2-2	11A6-2	10B11
						CT21	aa20-aa32	HTFPQTAIGVGAP-NH2	95％	98％	98％
CT22	aa24-aa32	QTAIGVGAP-NH2	92％	98％	98％
						CT23	aa27-aa32	IGVGAP-NH2	94％	97％	98％
CT24	aa28-aa32	GVGAP-NH2	95％	98％	98％
						CT25	aa29-aa32	VGAP-NH2	99％	93％	98％
CT26	aa30-aa32	GAP-NH2	37％	35％	60％

3.2.2 analysis of results:

epitope identification of the monoclonal antibody shows that the 8F6 epitope is mainly between aa14 and aa24, the 13A6 epitope and the 14D11 epitope are mainly between aa15 and aa21, and the 3A2 epitope is mainly between aa15 and aa 21. The 13C2-2, 11A6-2, 10B11 epitope is predominantly between aa29-aa 32.

The 3.2.3CT antigen is diluted by carbonic acid buffer solution (20 mmol/L CB, pH 9.6) and then coated on a polyvinyl chloride plate, seven antibodies purified by a protein A column are subjected to pretreatment and incubated with the synthesized CT alanine mutant polypeptide for 30min according to a certain proportion (the synthesized sequences are respectively supplemented at last) (50 ul of 100ng/ml antibody: 50ul of 50ug/ml alanine mutant polypeptide hapten), so as to obtain a detection sample; the control sample was incubated (50 ul of 100ng/ml antibody: 50ul of 20mM PBS) for 30min, the incubated test sample and control sample were then added to the CT antigen plate for 30min, the plate was washed 5 times, GAM-HRP (1/5000 dilution) was added for 30min, the plate was washed 5 times, and substrate was added for 15min. And finally, calculating and analyzing the detected OD value by reading the value under the wavelength of 450/620 of the enzyme label instrument, wherein a calculation formula is (OD value of a control sample-OD value of a detection sample)/OD value of the control sample, and the judgment standard is as follows: more than 90% showed good competition effect, showing that the amino acid was a key amino acid, and the data analysis and evaluation are as follows in tables 6 and 7.

TABLE 6 epitope result analysis

TABLE 7 epitope result analysis

Name of the name	Amino acid position	Amino acid sequence	13C2-2	11A6-2	10B11
						CT43	CT2432-1	ATAIGVGAP-NH2	92％	98％	97％
CT44	CT2432-2	QAAIGVGAP-NH2	94％	98％	98％
						CT45	CT2432-4	QTAAGVGAP-NH2	93％	97％	97％
CT46	CT2432-5	QTAIAVGAP-NH2	91％	95％	92％
						CT47	CT2432-6	QTAIGAGAP-NH2	56％	53％	85％
CT48	CT2432-7	QTAIGVAAP-NH2	70％	75％	62％
						CT49	CT2432-9	QTAIGVGAA-NH2	47％	36％	56％

3.2.4 analysis of results:

the key influencing sites of 8F6 are amino acids 15, 16, 17, 19, 20, 22, 23; the key influencing sites of 13A6, 14D11,3A2 are amino acids 16, 17, 19, 20; the key influencing sites of 13C2-2, 11A6-2, 10B11 are amino acids 29, 30, 31, 32; consistent with the results of epitope identification above.

Example 4 screening of magnetic bead coated mab and acridinium ester labeled mab in double antibody sandwich method

The optimal pairing of magnetic bead coated mab and acridinium ester labeled mab was screened experimentally using conventional double antibody sandwich assay conditions.

4.1 preparation of Callitonin magnetic bead coated mab:

preparing magnetic microparticle solution, wherein the magnetic microparticles are magnetic beads coated with hydrophilic polymer and carboxyl, and the particle size is 1.5-3 um; the preparation method comprises the following steps: adding 50mM MES solution with pH of 5.0 into magnetic microparticles and EDC and NHS at mass ratio of 1:1:1 to make magnetic microparticles have concentration of 4mg/mL, and placing on a vertical rotator for activation at 25deg.C for 20min; placing activated magnetic microparticles and anti-calciponin monoclonal antibody with the proportion of 15ug marked by each milligram of magnetic microparticles on a vertical rotator for marking, and reacting at the temperature of 25 ℃ for 3 hours; washing the reacted magnetic microparticles with washing liquid for 3 times, adding phosphate buffer solution containing glycine, 0.5% bovine serum albumin, 0.05% Triton X-100 and pH of 7.4 to make the concentration of the magnetic microparticles be 4mg/mL, and placing on a vertical rotator to terminate the reaction at the temperature of 25 ℃ for 2 hours; washing the magnetic microparticles after termination with washing solution for 3 times, adding phosphate buffer solution containing 0.5% (W/V) bovine serum albumin, 0.5% (W/V) casein, 0.05% T (W/V) riton X-100, antiseptic and pH 7.4 to make the concentration of magnetic microparticles be 4mg/mL, and preserving at 2-8deg.C for use;

The method is used for identifying the epitope in 28-32-NH ₂ The monoclonal antibodies (13C 2-2, 11A6-2, 10B 11) of amino acids (B) were subjected to MS300 bead coating to prepare magnetic microparticle solutions.

4.2 preparation of Calcitonin acridine ester labeled mab:

the preparation method of the acridinium ester labeled antibody solution comprises the following steps: taking 50ug of anti-Callitonin monoclonal antibody to be marked, adding phosphate buffer solution containing NaCl to a volume of 300 mu L, adding 5 mu L of acridinium ester mother solution, shaking and mixing uniformly, and reacting for 30min at room temperature in a dark place; after the reaction, 200 mu L of phosphate buffer solution containing NaCl and glycine is added, and the mixture is manually inverted for 20 times and uniformly mixed, and the mixture reacts for 30 minutes at room temperature in a dark place; transferring the product into a dialysis bag after the reaction, dialyzing the dialyzate into 20mM PBS buffer solution with pH of 7.4 at 2-8deg.C in the dark, and replacing the PBS buffer solution every 2h for 3 times to remove unlabeled acridine ester; taking out the marker, adding 10% (W/V) bovine serum albumin to the final concentration of 0.1% (V/V1:100) bovine serum albumin according to the actual volume, adding equal volume of glycerol, manually reversing and mixing, and preserving in dark below-15 ℃ for later use.

Acridine ester labeling was performed on 3 strains of Calcitonin monoclonal antibody (14D11,2E12, 13 A6) recognizing antibody epitopes at 15-21 amino acids in the manner described above.

4.3 experimental method:

4.3.1 specimens:

CT serum sample, collect Roche background serum sample, because CT serum sample is easy to degrade, so the preservation of serum sample is-20 degrees preserved, 4 degrees preserved and used up in 3 days. High value samples of 1 CT background and 20% nbs deployment detection were used.

4.3.2 sample addition:

sampling 50ul of samples, adding 50 magnetic bead coated mab, incubating for 15min at 37 ℃, washing with Tween 20 phosphate buffer containing 0.05-0.08% after incubation, adding 50ul of acridinium ester labeled mab, incubating for 10min at 37 ℃ after shaking and mixing uniformly, washing with Tween 20 phosphate buffer containing 0.05-0.08% after incubation, adding pre-excitation liquid for 100-200 ul, and pre-excitation. And removing the pre-excitation liquid, adding 100-200 ul of the excitation liquid, and performing excitation and detection.

The pairing of each magnetic bead coated monoclonal antibody and the acridinium ester marked monoclonal antibody is detected in an orthogonal manner by the method, and the P/N (the ratio of the detection mean value of positive samples to the detection mean value of 20% NBS) value is obtained. The results are shown in Table 8 below.

TABLE 9 monoclonal antibody pairing results

Analysis of results: based on the P/N results we pick pairs of P/N >100 for amplified sample correlation detection.

The 4 pairs of P/N >100 were tested for 16 samples in the manner described above, and the data were subjected to a fixed comparison of sensitivity and correlation with background reagents, the results being shown in Table 10 below.

TABLE 10 monoclonal antibody pairing test results

Analysis of results: by detecting 16 samples, the pair 10B11/14D11 is closest to the background value after detection of the fixed value, and the pair is further prepared into a chemiluminescent detection kit according to whether the pair is distinguished by samples with the concentration lower than 0.5 pg/mL.

Example 5: parallel evaluation by using self-made calcitonin chemiluminescent detection kit and control reagent (roche reagent) Estimated WHO calibrator

The WHO calibrator used in this example was WHO International Standard calcitonin (Human NIBSC code:89/620Instructions for use (Version 5.0, date 28/03/2013)).

5.1 preparation of detection kit

5.1.1 preparation of magnetic bead coated mab:

the magnetic microparticles are magnetic beads coated with hydrophilic polymer and carboxyl, and the particle size is 1.5-3 um; the preparation method comprises the following steps: adding 50mM MES solution with pH of 5.0 into magnetic microparticles and EDC and NHS at mass ratio of 1:1:1 to make magnetic microparticles have concentration of 4mg/mL, and placing on a vertical rotator for activation at 25deg.C for 20min; placing the activated magnetic microparticles and the Calcitonin monoclonal antibody with the proportion of 10B11 monoclonal antibody of 15ug marked per milligram of the magnetic microparticles on a vertical rotator for marking, wherein the reaction environment temperature is 25 ℃ for 3 hours; washing the reacted magnetic microparticles with washing liquid for 3 times, adding phosphate buffer solution containing glycine, 0.5% bovine serum albumin, 0.05% Triton X-100 and pH of 7.4 to make the concentration of the magnetic microparticles be 4mg/mL, and placing on a vertical rotator to terminate the reaction at the temperature of 25 ℃ for 2 hours; washing the magnetic microparticles after termination with washing solution for 3 times, adding phosphate buffer solution containing 0.5% (W/V) bovine serum albumin, 0.5% (W/V) casein, 0.05% T (W/V) riton X-100, antiseptic and pH 7.4 to make the concentration of magnetic microparticles 4mg/mL, and preserving at 2-8deg.C for use.

5.1.2 preparation of acridinium ester-labeled mab:

the preparation method comprises the following steps: taking 50ug of 14D11 monoclonal antibody to be marked, adding phosphate buffer solution containing NaCl to a volume of 300 mu L, adding 5 mu L of acridinium ester mother solution, shaking and mixing uniformly, and reacting for 30min at room temperature in a dark place; after the reaction, 200 mu L of phosphate buffer solution containing NaCl and glycine is added, and the mixture is manually inverted for 20 times and uniformly mixed, and the mixture reacts for 30 minutes at room temperature in a dark place; transferring the product into a dialysis bag after the reaction, dialyzing the dialyzate into 20mM PBS buffer solution with pH of 7.4 at 2-8deg.C in the dark, and replacing the PBS buffer solution every 2h for 3 times to remove unlabeled acridine ester; taking out the marker, adding 10% (W/V) bovine serum albumin to the final concentration of 0.1% (V/V1:100) bovine serum albumin according to the actual volume, adding equal volume of glycerol, manually reversing and mixing, and preserving in dark below-15 ℃ for later use.

5.2 detection method:

1. preparation: the kit obtained in 5.1 was allowed to stand at room temperature (18-30 ℃) for 15-30min. 2. Preparing liquid: 50ml of the concentrated washing solution (20X) was diluted to 1000ml with distilled or deionized water for use. 3. Sample adding: 50ul of the sample to be tested was added to each of the corresponding wells. 4. The reaction: adding 50ul of magnetic bead coated monoclonal antibody 10B11 into the sample hole, uniformly mixing, sealing by a sealing plate membrane, and incubating at 37+/-1 ℃ for 15min; after the incubation is finished for 15 to 20 minutes, washing with a Tween 20 phosphate buffer solution containing 0.05 to 0.08 percent, adding 50ul of acridinium ester labeled antibody 14D11, incubating for 10 to 15 minutes, after the incubation is finished, washing with a Tween 20 phosphate buffer solution containing 0.05 to 0.08 percent, adding 100 to 200ul of pre-excitation liquid, and pre-exciting. And removing the pre-excitation liquid, adding 100-200 ul of the excitation liquid, and performing excitation and detection. 5. The WHO standard was tested by a self-made calcitonin chemiluminescent kit. The detection results are shown in FIG. 1.

Analysis of results:

comparing the contrast luminescence values of the ROCHE reagent and the self-made chemiluminescent reagent of the present application using a CT WHO calibrator, we found that the luminescence values of the paired 10B11/14D11 of the present application were 13638, 7457, 4027, 2413, 1733 and 1096 when the CT (WHO) calibrator was used at 2pg/ml,1pg/ml,0.5pg/ml,0.25pg/ml,0.125pg/ml, and 0 pg/ml; while the luminescence values 2302, 1799, 1527, 1388, 1365 and 1297 of the Roche reagents; from the detection of CT (WHO) calibrator, it was found that the antibody of the application had a gradient change in the gradient diluted luminescence value of 2pg/ml, but the luminescence value of Roche reagent had no gradient change, and we considered that our chemiluminescent reagent had higher sensitivity than Roche reagent.

Example 6: assessment of low value samples using self-made calcitonin chemiluminescent assay kit

We further evaluated more samples below 0.5pg/mL for this pair. 140 samples of less than 0.5pg/mL were assayed for pairing of each bead coated mab and acridinium ester-labeled mab using the method described above in quadrature, with the results shown in Table 11 below.

TABLE 11 detection results

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Analysis of results: the samples with the paired 10B11/14D11 detection control reagents of less than 0.5pg/mL also tend to be used. For samples with a portion of the control reagent less than 0.5pg/mL, the detection of the luminescent reagent higher than 0.5pg/mL is explained by the phenomenon that the lower limit of the detection of the control reagent is insufficient, and further the sensitivity of the detection of the calcitonin target in human body is higher.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

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Claims

1. An antibody or antigen-binding fragment thereof that specifically binds Calcitonin (CT), comprising:

and/or the number of the groups of groups,

(vi) VL CDR3 consisting of the sequence: SEQ ID NO. 6, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto;

preferably, the substitutions described in any one of (i) - (vi) are conservative substitutions.

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

(i) SEQ ID NO: 7;

and

(iv) SEQ ID NO: 8;

(vi) And SEQ ID NO:8, a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

preferably, the substitution set forth in (ii) or (v) is a conservative substitution;

Preferably, the antibody or antigen binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO. 7 and a VL comprising the sequence shown as SEQ ID NO. 8.

3. An antibody or antigen-binding fragment thereof that specifically binds Calcitonin (CT), comprising:

and/or the number of the groups of groups,

(vi) VL CDR3 consisting of the sequence: 15, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto;

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

(i) SEQ ID NO:16, a sequence shown in seq id no;

and

(iv) SEQ ID NO:17, a sequence shown in seq id no;

(vi) And SEQ ID NO:17, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

preferably, the antibody or antigen binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO. 16 and a VL comprising the sequence shown as SEQ ID NO. 17.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, which is murine or humanized;

Preferably, the antibody or antigen binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin;

preferably, the antibody or antigen binding fragment thereof comprises a constant region derived from a human immunoglobulin;

preferably, the heavy chain of the antibody or antigen binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., igG1, igG2, igG3 or IgG 4), and/or the light chain of the antibody or antigen binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (e.g., kappa or lambda).

6. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-5, or a heavy chain variable region and/or a light chain variable region thereof.

7. A vector comprising the nucleic acid molecule of claim 6; preferably, the vector is a cloning vector or an expression vector.

8. A host cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7.

9. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-5, comprising culturing the host cell of claim 8 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

10. A conjugate comprising the antibody or antigen-binding fragment thereof of claim 1 or 2, and a detectable label attached to or coated with the antibody or antigen-binding fragment thereof;

preferably, the detectable label is selected from the group consisting of enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters, luminol and derivatives thereof, or ruthenium derivatives), fluorescent dyes (e.g., fluorescein or fluorescent protein), radionuclides, biotin, or magnetic beads (e.g., amino-bearing immunomagnetic beads, carboxyl-bearing immunomagnetic beads, hydroxyl-bearing immunomagnetic beads, aldehyde-bearing immunomagnetic beads);

preferably, the detectable label is a magnetic bead.

11. A conjugate comprising the antibody or antigen-binding fragment thereof of claim 3 or 4, and a detectable label attached to or coated with the antibody or antigen-binding fragment thereof;

Preferably, the detectable label is an acridine ester compound.

12. A kit, comprising: (i) a first antibody or first conjugate, or (ii) a second antibody or second conjugate, or (iii) a combination thereof;

the first antibody is the antibody or antigen-binding fragment thereof of claim 1 or 2, and the first conjugate is the conjugate of claim 10;

the second antibody is an antibody capable of binding to a different epitope of Calcitonin (CT); the second conjugate comprises a second antibody and a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound, luminol and derivatives thereof, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin; preferably, the second conjugate comprises a second antibody and an acridinium ester;

preferably, the second antibody is capable of binding amino acids 16, 17, 19 and/or 20 of Calcitonin (CT);

more preferably, the second antibody is the antibody or antigen binding fragment thereof of claim 3 or 4;

preferably, the second conjugate is a conjugate according to claim 11.

13. A method for detecting the presence or level of calcitonin in a biological sample, the method comprising:

comprising the use of an antibody or antigen-binding fragment thereof according to any one of claims 1-5 or a conjugate according to claim 10 or 11 or a kit according to claim 12;

preferably, the method is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay;

preferably, the method comprises: contacting the sample with a first antibody or first conjugate that binds to a different epitope of a calcitonin antigen, and a second antibody or second conjugate, and qualitatively or quantitatively detecting binding of the two antibodies to the calcitonin antigen, wherein binding is indicative of the presence or concentration of the calcitonin antigen in the sample;

preferably, the first antibody is the antibody or antigen-binding fragment thereof of claim 1 or 2;

preferably, the first conjugate is a conjugate according to claim 10;

preferably, the second antibody is the antibody or antigen binding fragment thereof of claim 3 or 4;

preferably, the second conjugate is a conjugate according to claim 11.

14. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-5 or the conjugate of claim 10 or 11 in the manufacture of a kit for detecting the presence or level of calcitonin in a sample and/or for diagnosing whether a subject has a disease caused by altered calcitonin levels (e.g. osteoporosis, medullary thyroid cancer, lung cancer, small cell lung cancer, breast cancer, tumors of neural kurtosis origin, hyperthyroidism, hypergastrinemia, renal failure and chronic inflammatory disease);

preferably, the kit is a kit according to claim 12;

preferably, the kit detects the presence or level of calcitonin in a sample by the method of claim 13;

preferably, the sample is a blood sample (e.g., whole blood, plasma or serum), fecal matter, oral or nasal secretions, or alveolar lavage from a subject (e.g., a mammal, preferably a human);

preferably, the calcitonin has the sequence shown in SEQ ID NO. 9.