Anti-BSA antibodies and their applications

Abstract

The present invention relates to an anti-BSA antibody and its application, in particular to a specific antibody targeting bovine serum albumin (BSA) and not binding to human serum albumin (HSA). The antibody of the present invention has affinity, stability and specificity. The antibody of the present invention has broad application prospects in biomedical research, immune detection, clinical diagnosis and treatment. The present invention also provides a preparation method of the antibody and a specific application method thereof in related fields.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to highly specific anti-BSA antibodies and their applications in immunoassays, biomedicine, and other fields. Background Art

[0002] Bovine serum albumin (BSA), a protein found extensively in bovine serum, is often used as a standard and carrier in biopharmaceutical research and experiments. BSA is also a commonly used material in cell culture and is widely used in the biopharmaceutical field as a production process additive. Although most BSA can be removed from products through purification during the production process, trace amounts of BSA still exist in finished biologics. If introduced into the human body, this can cause severe allergic reactions and compromise the safety of biologics.

[0003] Therefore, the detection of BSA residual content is an important indicator of quality control of biological products and is directly related to the safety of biological products. The World Health Organization and pharmacopoeias of various countries in the world have strict limits on the residual BSA content. The 2020 edition of the "Chinese Pharmacopoeia" (Part III) clearly stipulates the residual amount of BSA in various vaccine drugs: the stock solution should not be higher than 50ng / mL (except for inactivated hepatitis A vaccine), and the finished product should not be higher than 50ng / dose. In addition, the actual detection of BSA is easily interfered with by HSA in the culture medium components. Therefore, this field urgently needs to develop a BSA antibody that has both high affinity and high specificity. Summary of the Invention

[0004] The object of the present invention is to provide a BSA antibody that has both high affinity for BSA and high specificity, thereby being able to distinguish BSA from other interfering substances.

[0005] Another object of the present invention is to provide uses of the BSA antibody of the present invention in the fields of immunoassay and even biomedicine.

[0006] In a first aspect, the present invention provides an antibody targeting BSA, wherein the antibody is selected from the group consisting of:

[0007] (1) An antibody comprising a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 13, and / or comprises HCDR2 as shown in SEQ ID NO: 14, and / or comprises HCDR3 as shown in either SEQ ID NO: 15 or SEQ ID NO: 19;

[0008] (2) An antibody comprising a light chain variable region, wherein the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 16, and / or comprises LCDR2 as shown in SEQ ID NO: 17, and / or comprises LCDR3 as shown in either SEQ ID NO: 18 or SEQ ID NO: 20;

[0009] (3) an antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody;

[0010] (4) An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region CDR of the antibody has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the heavy chain variable region CDR of the antibody described in (1), (2) or (3) above, and / or the light chain variable region CDR of the antibody has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the light chain variable region CDR of the antibody described in (1), (2) or (3) above.

[0011] In a preferred embodiment, the CDRs of the antibody are determined according to the Kabat numbering convention.

[0012] In a specific embodiment, the antibody is selected from:

[0013] (1) An antibody comprising HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 15, LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 17, and LCDR3 shown in SEQ ID NO: 18;

[0014] (2) An antibody comprising HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 19, LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 17, and LCDR3 shown in SEQ ID NO: 20;

[0015] (3) An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region CDR of the antibody has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the heavy chain variable region CDR of the antibody described in (1) or (2) above, and the light chain variable region CDR of the antibody has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the light chain variable region CDR of the antibody described in (1) or (2) above.

[0016] In a specific embodiment, the antibody is selected from:

[0017] (1) An antibody, wherein the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 3;

[0018] (2) An antibody, wherein the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4;

[0019] (3) an antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody;

[0020] (4) An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region of the antibody has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the heavy chain variable region of the antibody described in (1), (2) or (3) above, and / or the light chain variable region of the antibody has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the light chain variable region of the antibody described in (1), (2) or (3) above.

[0021] In a specific embodiment, the antibody is selected from:

[0022] (1) An antibody, wherein the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 2;

[0023] (2) An antibody, wherein the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 3 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 4;

[0024] (3) An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region CDR of the antibody has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the heavy chain variable region CDR of the antibody described in (1) or (2) above, and the light chain variable region CDR of the antibody has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the light chain variable region CDR of the antibody described in (1) or (2) above.

[0025] In a specific embodiment, the antibody is selected from:

[0026] (1) An antibody comprising a heavy chain and a light chain, wherein the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO: 5, and the light chain of the antibody has the amino acid sequence shown in SEQ ID NO: 6;

[0027] (2) An antibody comprising a heavy chain and a light chain, wherein the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO: 7, and the light chain of the antibody has the amino acid sequence shown in SEQ ID NO: 8;

[0028] (3) An antibody comprising a heavy chain and a light chain, wherein the heavy chain of the antibody has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the heavy chain of the antibody described in (1) or (2) above, and the light chain of the antibody has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the light chain of the antibody described in (1) or (2) above.

[0029] In a preferred embodiment, the antibody recognizes only BSA and not HSA.

[0030] In a second aspect, the present invention provides a nucleic acid encoding the antibody of the first aspect.

[0031] In a third aspect, the present invention provides an expression vector comprising the nucleic acid described in the second aspect.

[0032] In a fourth aspect, the present invention provides a host cell comprising the expression vector described in the third aspect or a host cell in which the nucleic acid described in the second aspect is integrated into its genome.

[0033] In a fifth aspect, the present invention provides an immunoconjugate comprising:

[0034] 1) the antibody of the first aspect; and

[0035] 2) Detectable label.

[0036] In a preferred embodiment, the detectable label is a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computer tomography) contrast agent, or an enzyme or radionuclide capable of producing a detectable product.

[0037] In a sixth aspect, the present invention provides a detection kit for BSA detection, the detection kit comprising:

[0038] 1) The antibody according to the first aspect;

[0039] 2) container; and

[0040] 3) Instructions for use that guide the user on how to use the antibody to detect BSA in a sample to be tested.

[0041] In a seventh aspect, the present invention provides a method for detecting BSA, comprising the following steps:

[0042] 1) contacting the sample to be tested with the antibody of the first aspect; and

[0043] 2) Detecting whether a complex between BSA and the antibody is formed in the sample to be tested. If a complex is formed, it indicates that BSA is present in the sample.

[0044] In an eighth aspect, the present invention provides use of the antibody described in the first aspect in preparing a detection reagent or a detection kit for BSA.

[0045] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Monoclonal ELISA results are shown (plate 1);

[0047] Figure 2 Monoclonal ELISA results are shown (plate 2);

[0048] Figure 3 Monoclonal ELISA results are shown (plate 3);

[0049] Figure 4 Monoclonal ELISA results are shown (plate 4);

[0050] Figure 5 Specific identification results are shown;

[0051] Figure 6 The alignment results of anti-BSA antibody sequences are shown;

[0052] Figure 7 SDS PAGE characterization of the 1G3 full-length antibody is shown;

[0053] Figure 8 The EC of 1G3 full-length antibody binding to BSA is shown. 50 Value determination;

[0054] Figure 9 The results of HRP-labeled antibody activity detection are shown. DETAILED DESCRIPTION

[0055] After extensive and in-depth research, the inventors unexpectedly discovered a highly effective anti-BSA antibody. This antibody, produced through genetic engineering techniques, exhibits high affinity, stability, and specificity. Specifically, the present invention utilizes genetic recombination technology to insert the gene sequence for a BSA-specific antibody into an expression vector, and then achieves efficient expression of the antibody through cell culture techniques. The resulting antibody, after purification, exhibits high affinity and stability, specifically recognizing and binding to BSA. This is the basis for the present invention.

[0056] Definition of terms

[0057] The terms used herein have the same or similar meanings as those commonly understood by those skilled in the art. For the sake of clarity, some of the terms are defined below.

[0058] The term "bovine serum albumin (BSA)" refers to a globulin found in bovine serum, also known as the fifth fraction. This protein contains 583 amino acid residues, has a molecular weight of 66.430 kDa, and an isoelectric point of 4.7. BSA is widely used in biochemical experiments. It's worth noting that the fifth fraction isn't a component of BSA, but rather one of five components that Professor Edwin J. Cohn isolated from blood through a specific separation technique. The fifth of these components is albumin.

[0059] Human serum albumin (HSA) is a highly water-soluble, globular, monomeric plasma protein. It is synthesized by hepatocytes and accounts for approximately 50% of the total protein synthesized by the liver. HSA is a non-glycosylated single-chain polypeptide consisting of 585 amino acids with a molecular weight of approximately 66 kDa. The protein exhibits a heart-shaped structure with three homologous regions and a complex substructure and helical structure.

[0060] The term "horseradish peroxidase (HRP)" refers to a heme-containing protein with a molecular weight of approximately 40,000 and an isoelectric point of 7.2. It is readily soluble in water and exhibits high specific activity, stability, and a low molecular weight, making the preparation of pure enzymes easy. Horseradish peroxidase is a catalytic enzyme with a cooperative structure that produces the oxygen and electron donors required for the catalytic environment during the catalytic process. Horseradish peroxidase is also an important tool enzyme in immunology and clinical chemistry. Its high purity enables its application in immunological reactions, such as enzyme-labeled immunoassays in conjunction with specific antigens to measure the content of various biomolecules.

[0061] The term "antibody" as used herein refers to an antigen-binding protein of the immune system. As referred to herein, the term "antibody" includes intact, full-length antibodies with an antigen-binding region, as well as any fragments thereof in which the "antigen-binding portion" or "antigen-binding region" is retained, or single chains thereof, such as single-chain variable fragments (scFv). A native antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding fragments thereof. The term "antibody" also includes all recombinant forms of antibodies (particularly those described herein), such as those expressed in prokaryotic cells, non-glycosylated antibodies, and antigen-binding antibody fragments and derivatives described below. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). VH and VL can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL chain is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen.

[0062] Antibody fragments include, but are not limited to: (i) Fab fragments consisting of the VL, VH, CL and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments consisting of the VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region (Ward et al., 1989, Nature 341:544-546); (v) F(ab')2 fragments, bivalent fragments comprising two linked Fab fragments; (vi) single-chain Fv molecule antigen-binding sites (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883); (vii) bispecific single-chain Fv dimers (PCT / US92 / 09965); (viii) "dibodies" or "tribodies," multivalent or multispecific fragments constructed by genetic fusion (Tomlinson et al., 2000, Methods Enzymol. 326:461-479; WO 94 / 13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448); and (ix) scFv genetically fused to the same or different antibodies (Coloma & Morrison, 1997, Nature Biotechnology 15, 159-163).

[0063] The term "Fc" or "Fc region" includes polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-termini of these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc includes the immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2.

[0064] All immunoglobulin heavy chain constant region positions discussed herein are numbered according to the EU index as in Kabat (Kabat et al., 1991, sequences of proteins of immunological interest, 5th ed., United States Public Health Service, National Institutes of Health, Bethesda, incorporated by reference in its entirety). "Kabat's EU index" refers to the residue numbering of the human IgG1 EU antibody as described by Edelman et al., 1969, Biochemistry 63:78-85.

[0065] Antibodies of the present invention

[0066] The present invention provides a highly effective anti-BSA antibody having high affinity, stability and specificity.

[0067] The present invention provides antigen-binding proteins, including antibodies, having scFv-based antigen-binding regions. Specifically, the inventors selected scFvs from a human scFv phage display library. In addition to high affinity for BSA, the antibodies of the present invention exhibit exquisite specificity. For example, the antibodies of the present invention only recognize BSA, but not HSA.

[0068] In a specific embodiment, the antibodies of the present invention are clones 1C3 and 2G2, and the antibody variable region sequences of the two are shown in Table 1:

[0069] Table 1 Variable region sequences of monoclonal antibodies against BSA

[0070]

[0071]

[0072] According to the Kabat numbering rules, the CDR regions corresponding to these two antibodies are shown in Table 2:

[0073] Table 2 CDR region sequences of monoclonal antibodies against BSA

[0074]

[0075] In some embodiments, the present invention includes antibodies having scFv sequences fused to one or more heavy chain constant regions to form antibodies having human or mouse or other species immunoglobulin Fc regions to produce bivalent proteins, thereby increasing the overall affinity and stability of the antibody. In addition, the Fc portion allows other molecules (including but not limited to fluorescent dyes, horseradish peroxidase (HRP), radioisotopes, etc.) to be directly conjugated to antibodies for use in, for example, antigen quantification studies.

[0076] In one embodiment, the anti-BSA antibody is a full-length antibody, that is, the heavy chain and light chain of the antibody can be full-length (for example, the antibody can include at least one and preferably two complete heavy chains, and at least one and preferably two complete light chains). In other embodiments, the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE. The choice of antibody type will depend on the immune effector function that the designed antibody is intended to elicit. When constructing recombinant immunoglobulins, suitable amino acid sequences of the constant regions of various immunoglobulin isotypes and methods for producing a wide range of antibodies are known to those skilled in the art. The antibody variable regions of antibodies 1C3 and 2G2 are fused with the constant region of a mouse antibody to form the full-length antibody amino acid sequence shown in Table 3, and the nucleotide sequence shown in Table 4.

[0077] Table 3 Amino acid sequences of full-length antibodies against BSA

[0078]

[0079]

[0080] Table 4 Nucleotide sequences of full-length antibodies against BSA

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] The anti-BSA antibodies of the present invention can be used in a variety of fields, including biomedical research, immunoassays, and clinical diagnosis and treatment. For example, the anti-BSA antibodies of the present invention can be used to study the structure and function of BSA, providing a powerful tool for research in the biomedical field. The anti-BSA antibodies of the present invention can be used in immunoassays. For example, the antibodies can be used to construct highly sensitive immunoassay methods to achieve quantitative and qualitative detection of BSA. The anti-BSA antibodies of the present invention can also be used in clinical diagnosis and treatment. The antibodies can be used to prepare immunodiagnostic reagents and immunotherapeutic drugs, providing new means for diagnosing and treating related diseases.

[0087] Therefore, to apply the antibodies of the present invention to the aforementioned fields, the present invention provides an immunoconjugate comprising the antibodies of the present invention and a detectable label. The detectable label includes, but is not limited to, a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme or radionuclide capable of producing a detectable product. The present invention also provides a test kit for detecting BSA, comprising the antibodies of the present invention, a container, and instructions for use instructing the user to use the antibodies to detect BSA in a test sample.

[0088] The present invention also provides a method for detecting BSA, comprising contacting a sample to be tested with the antibody of the present invention; and then detecting whether a complex between BSA and the antibody is formed in the sample to be tested. If a complex is formed, it indicates the presence of BSA in the sample.

[0089] Based on the teachings of the present invention and conventional techniques in the art, those skilled in the art will know how to obtain the antibodies of the present invention. In a specific embodiment, the present invention provides a method for preparing the anti-BSA antibody of the present invention, comprising steps such as gene cloning, vector construction, cell culture, antibody expression, and purification.

[0090] Nucleic acids, vectors and host cells

[0091] To obtain the anti-BSA antibodies of the present invention, the present invention also provides nucleic acids encoding antibodies that bind to BSA and fragments thereof, vectors, and host cells containing the nucleic acids or vectors. The nucleic acids can be in intact cells, in cell lysates, or in partially purified or substantially purified form.

[0092] The nucleic acids of the present invention can be obtained using standard molecular biology techniques, for example, cDNA encoding the light and heavy chains of an antibody or encoding the VH and VL segments can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), one or more nucleic acids encoding the antibody can be recovered from the library. Methods for introducing exogenous nucleic acids into host cells are generally known in the art and can vary depending on the host cell used.

[0093] In a specific embodiment, the nucleic acid molecule of the present invention is a nucleotide sequence encoding a light chain variable region selected from SEQ ID NOs: 10 and 12 and / or a nucleotide sequence encoding a heavy chain variable region selected from SEQ ID NOs: 9 and 11. In a preferred embodiment, the nucleic acid molecule of the present invention comprises a sequence of SEQ ID NO: 9 encoding the heavy chain of an antibody of the present invention and a sequence of SEQ ID NO: 10 encoding the light chain of an antibody of the present invention, or comprises a sequence of SEQ ID NO: 11 encoding the heavy chain of an antibody of the present invention and a sequence of SEQ ID NO: 12 encoding the light chain of an antibody of the present invention.

[0094] In order to express proteins, nucleic acids encoding antibodies of the present invention can be integrated into expression vectors. A variety of expression vectors can be used for protein expression. Expression vectors can include self-replicating extrachromosomal vectors, or vectors that integrate into the host genome. Expression vectors used in the present invention include, but are not limited to, those that enable protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. As is known in the art, a variety of expression vectors are commercially available or otherwise obtainable. Can be used in the present invention to express antibodies.

[0095] Advantages of the present invention:

[0096] 1. The anti-BSA antibody of the present invention has a high affinity for BSA;

[0097] 2. The anti-BSA antibody of the present invention has excellent stability;

[0098] 3. The anti-BSA antibody of the present invention only recognizes BSA, not HSA, and therefore has excellent specificity;

[0099] 4. The anti-BSA antibody of the present invention provides a new material means for many fields such as biomedical research, immunoassay, clinical diagnosis and treatment.

[0100] The following specific examples further illustrate the present invention. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (Molecular Cloning-A Laboratory Manual) (3rd Edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise stated.

[0101] Example 1. Screening of antibodies against BSA using a phage library

[0102] The phage display library used in this invention is a fully human scFv phage library constructed by our company with a library capacity of 2E+10. A highly specific scFv antibody against BSA was obtained by biopanning. Briefly, 10 μg / ml BSA (Sigma, # A1933 The phage library was added to a BSA-coated immunotube and allowed to bind for 1.5 hours. Nonspecific phage was then washed away, and bound phage was eluted and used to infect E. coli TG1 cells in logarithmic growth phase. The eluted phage was expanded and purified using PEG / NaCl precipitation for the next round of screening. Panning was performed for 3-4 cycles to enrich for scFv phage clones that specifically bound to BSA.

[0103] Example 2. Identification of monoclonal antibodies against BSA using ELISA

[0104] After the fourth round of screening, monoclonal clones were picked into 96-well plates, and the antibody was induced to express in the culture supernatant using IPTG. A total of 4 96-well plates were picked and named Plate 1-4. BSA was diluted to 5 μg / ml with PBS, and 100 μL / well was added to the enzyme-labeled plate and incubated overnight at 4 degrees; blocked with 2% skim milk powder at room temperature for 2 hours; 50 μl / well induced supernatant + 50 μl / well 4% skim milk powder were added and incubated at room temperature for 1 hour; washed three times with PBS, and then 100 μl / well of anti-Flag tag antibody diluted 1:4000 was added and incubated at room temperature for 1 hour; washed three times with PBS, and 100 μl / well TMB solution was added for color development. After color development for 10 minutes, 50 μl / well 1M H2SO4 was added to terminate the reaction. The absorbance value of each well was measured using an enzyme-labeled instrument to determine the OD value. 450nM The results are as follows Figure 1-4 As shown, each plate has more than 40 positive clones (OD 450nM >0.5), with a total of 149 positive clones across four plates.

[0105] Example 3. Specific identification of monoclonal antibodies against BSA using ELISA

[0106] Pick the 24 best candidate clones from the initial screening and repeat the induction, and identify the specificity by ELISA. The specific description is as follows: dilute BSA or HSA (Sigma, #A9511) with PBS to 5μg / ml, add 100μL / well to the enzyme-labeled plate, and incubate at 4 degrees overnight; block with 2% skim milk powder at room temperature for 2 hours; add 50μl / well induction supernatant + 50μl / well 4% skim milk powder, incubate at room temperature for 1 hour; wash three times with PBS, then add 100μl / well, 1:4000 diluted anti-Flag tag antibody, incubate at room temperature for 1 hour; wash three times with PBS, add 100μl / well TMB solution for color development, after 10 minutes of color development, add 50μl / well 1M H2SO4 to terminate the reaction. Use an enzyme-labeled instrument to measure the absorbance value OD of each well. 450nM The results are as follows Figure 5 As shown, each clone specifically binds to BSA but not to HSA, wherein a control antibody was obtained by the inventors through a conventional hybridoma method, and the control antibody binds to both BSA and HSA.

[0107] Example 4. Sequencing of monoclonal antibodies specific for BSA

[0108] Candidate clones that specifically bind to BSA were selected for sequencing. A total of two antibodies with unique sequences, 1C3 and 2G2, were obtained. The variable region sequences of their light and heavy chains are shown in Figure 2. Figure 6 shown.

[0109] Example 5. Construction of full antibody

[0110] The heavy chain variable region of the candidate clone 1C3 was fused with the heavy chain constant region of mouse IgG1 (reference sequence, uniprot P01868) to form the heavy chain sequence of the full antibody. The light chain variable region of the candidate clone 1C3 was fused with the mouse kappa chain constant region (reference sequence, uniprot P01837) to form the light chain of the full antibody. The amino acid sequence of the heavy chain or light chain full antibody was reverse translated into a nucleotide sequence. The specific sequence information is shown in Table 3. The full-length sequence of the 1G3 light chain and heavy chain was constructed into the eukaryotic expression plasmid PTT5. The two plasmids were then transiently transfected into CHO-S cells for expression, and the expression product was affinity purified by protein A and then identified for purity by SDS-PAGE. The results are shown in Table 3. Figure 7 The EC50 value of 1C3 full-length antibody binding to BSA was detected by ELISA, and the results are shown in Figure 8 As shown, the EC of 1C3 full-length antibody binding to BSA 50 The value was 0.87 μg / mL.

[0111] Example 6.1 HRP labeling and activity identification of G3 complete antibody

[0112] Antibodies were HRP-labeled using the periodate method. The HRP labeling kit (Frdbio, ARL0010K) was removed from 4°C and equilibrated at room temperature for 30 minutes to allow all components to fully thaw and mix. To 100 μg of antibody solution, 1 / 10 volume of labeling buffer was added. Mix thoroughly by pipetting several times to avoid bubbles. Add an equal amount of horseradish peroxidase to the target material and mix thoroughly by pipetting several times to avoid bubbles. Incubate at room temperature in the dark for 2 hours.

[0113] Then, add 1 μL of reaction stop solution per 10 μL of reaction solution. Mix thoroughly and let stand at room temperature for 1 hour. After termination, add an equal volume of marker storage solution, mix thoroughly, and store at -20°C.

[0114] Direct ELISA method to detect the activity of 1G3-HRP labeled antibody: dilute BSA to 5μg / mL with PBS, add 100μL / well to the enzyme-labeled plate, incubate at 4 degrees overnight; block with 2% gelatin at room temperature for 2 hours; wash three times with PBST, dilute 1G3-HRP 3-fold starting from 1:100 times with PBS 7 times, 100μL / well, incubate at room temperature for 1 hour; wash three times with PBST, add 100μL / well TMB solution for color development, color development for 20 minutes, add 50μl / well 1M H2SO4 to terminate the reaction. Use a microplate reader to measure the absorbance value of each well OD 450nM The results are as follows Figure 9 As shown, the 1G3 antibody can be directly used in BSA binding detection after being labeled with HRP, with a titer of 1:24300.

[0115] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An antibody targeting BSA, characterized in that: The antibody is selected from the group consisting of: (1) An antibody comprising HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 15, LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 17, and LCDR3 shown in SEQ ID NO: 18; (2) An antibody comprising HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 19, LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 17, and LCDR3 shown in SEQ ID NO:

20.

2. The antibody according to claim 1, characterized in that The CDRs of the antibodies were determined according to the Kabat numbering convention.

3. The antibody according to claim 1, characterized in that The antibody is selected from: (1) An antibody, wherein the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 2; (2) An antibody, wherein the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 3 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:

4.

4. The antibody according to claim 3, characterized in that The antibody is selected from: (1) An antibody comprising a heavy chain and a light chain, wherein the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO:5, and the light chain of the antibody has the amino acid sequence shown in SEQ ID NO:6; (2) An antibody comprising a heavy chain and a light chain, wherein the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO:7, and the light chain of the antibody has the amino acid sequence shown in SEQ ID NO:

8.

5. The antibody according to any one of claims 1 to 4, characterized in that The antibody only recognizes BSA and not HSA.

6. A nucleic acid encoding the antibody according to any one of claims 1 to 5. An expression vector comprising the nucleic acid according to claim 6 .

8. A host cell comprising the expression vector of claim 7 or the nucleic acid of claim 6 integrated into its genome.

9. An immunoconjugate comprising: 1) The antibody of any one of claims 1 to 5; and 2) Detectable markers.

10. The immunoconjugate according to claim 9, characterized in that The detectable label is selected from: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes and radionuclides capable of producing detectable products.

11. A detection kit for BSA detection, comprising: 1) The antibody according to any one of claims 1 to 5; 2) container; and 3) Instructions for use to guide users on how to use the antibody to detect BSA in a sample to be tested.

12. A method for detecting BSA, comprising the following steps: 1) contacting a sample to be tested with the antibody described in any one of claims 1 to 5; and 2) detecting whether a complex of BSA and the antibody is formed in the sample to be tested, and if a complex is formed, it indicates that BSA exists in the sample; The methods described should not be used for diagnostic or therapeutic purposes.

13. Use of the antibody according to any one of claims 1 to 5 in the preparation of a detection reagent or a detection kit for BSA.

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