Monoclonal antibodies against human sCD14-ST protein and their applications

Abstract

This invention discloses a monoclonal antibody against human sCD14-ST protein, relating to the field of diagnostic technology for human secretory sCD14. The monoclonal antibody was screened to yield six antibodies: Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6, whose heavy and light chain amino acid sequences are shown in SEQ ID NO. 85-96. These monoclonal antibodies exhibit higher specificity, affinity, and sensitivity against human sCD14-ST protein, enabling precise quantification of the protein. They can play an important role in diagnostic and prognostic detection, as well as in the study of the dynamic mechanism of human sCD14-ST protein in the field of immunology.

Description

Technical Field

[0001] This invention relates to the field of diagnostic technology for human secretory sCD14, and particularly to monoclonal antibodies against human sCD14-ST protein and their applications. Background Technology

[0002] Human CD14 protein is classified into two types: membrane-bound (mCD14) and secretory (sCD1). mCD14 is expressed in macrophages, neutrophils, and dendritic cells, and its main function is as a co-receptor for bacterial lipopolysaccharide (LPS) and other pathogen-associated molecules. Secretory sCD14 is further divided into high molecular weight (49KD-55KD) sCD14 and low molecular weight (11KD-15KD) sCD14-ST. While high molecular weight sCD14 is widely present in the blood of patients with sepsis, AIDS, and lupus erythematosus, it is not a specific molecular marker. sCD14-ST is composed of the N-terminus of high molecular weight sCD14, with a significant deletion of the C-terminal amino acids. Therefore, sCD14-ST and high molecular weight sCD14 have different immunogenicity and can be distinguished by different antibodies. Studies have found that sCD14-ST can serve as a sepsis-specific biological marker with potential biological value, thus attracting significant clinical attention. The consensus in treating sepsis is early identification and effective anti-infective therapy. Therefore, developing highly sensitive and specific antibodies that recognize sCD14-ST is of great significance for the early diagnosis of sepsis. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a monoclonal antibody against human sCD14-ST protein. Utilizing rabbit single-cell B-cell antibody development technology, sCD14-ST-specific single-cell B cells are isolated from the peripheral blood of rabbits immunized with sCD14-ST. Through PCR amplification and sequencing analysis, multiple antibodies specifically recognizing the anti-sCD14-ST protein are obtained. The monoclonal antibodies screened by this invention exhibit higher affinity and specificity compared to antibodies screened using traditional hybridoma technology and rabbit polyclonal antibodies; this increases the diversity of antibodies against sCD14-ST protein, laying a solid foundation for the development of highly sensitive sCD14-ST ELISA diagnostic kits. Specifically, this is achieved through the following techniques.

[0004] A monoclonal antibody against human sCD14-ST protein, including any one of Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6;

[0005] The amino acid sequences of the three complementary determinant regions CDR1, CDR2 and CDR3 of the heavy chain variable region of Ab1 are as shown in SEQ ID NO. 1-3 or the amino acid sequences with conserved modifications thereof, and the amino acid sequences of the three complementary determinant regions CDR1, CDR2 and CDR3 of the light chain variable region of Ab1 are as shown in SEQ ID NO. 4-6 or the amino acid sequences with conserved modifications thereof.

[0006] The amino acid sequences of the three complementarity-determining regions CDR1 and CDR3 of the heavy chain variable region of Ab2 are as shown in SEQ ID NO.7-8 or are amino acid sequences that have been conserved. The amino acid sequence of CDR2 is STG or is an amino acid sequence that has been conserved. The amino acid sequences of the three complementarity-determining regions CDR1, CDR2 and CDR3 of the light chain variable region of Ab2 are as shown in SEQ ID NO.9-11 or are amino acid sequences that have been conserved.

[0007] The CDR3 of the heavy chain variable region of Ab1 has the same amino acid sequence as the CDR3 of the heavy chain variable region of Ab2.

[0008] The amino acid sequences of the three complementarity-determining regions CDR1 and CDR3 of the heavy chain variable region of Ab3 are as shown in SEQ ID NO.12-13 or are conserved amino acid sequences thereof, and the amino acid sequence of CDR2 is SSD or is a conserved amino acid sequence thereof; the amino acid sequences of the three complementarity-determining regions CDR1, CDR2 and CDR3 of the light chain variable region of Ab3 are as shown in SEQ ID NO.14-16 or are conserved amino acid sequences thereof.

[0009] The amino acid sequences of the three complementarity-determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region of Ab4 are as shown in SEQ ID NO.17 or are conserved amino acid sequences thereof; the amino acid sequence of CDR2 is DSG or is a conserved amino acid sequence thereof; and the amino acid sequence of CDR3 is GDL or is a conserved amino acid sequence thereof. The amino acid sequences of the three complementarity-determining regions (CDR1, CDR2, and CDR3) of the light chain variable region of Ab4 are as shown in SEQ ID NO.18-20 or are conserved amino acid sequences thereof.

[0010] The CDR2 of the light chain variable region of Ab1 has the same amino acid sequence as the CDR2 of the light chain variable region of Ab4.

[0011] The amino acid sequences of the three complementary determinant regions CDR1, CDR2 and CDR3 of the heavy chain variable region of Ab5 are as shown in SEQ ID NO. 21-23 or the amino acid sequences with conserved modifications thereof, and the amino acid sequences of the three complementary determinant regions CDR1, CDR2 and CDR3 of the light chain variable region of Ab5 are as shown in SEQ ID NO. 24-26 or the amino acid sequences with conserved modifications thereof.

[0012] The amino acid sequences of the three complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of Ab6 are shown in SEQ ID NO. 27-29 or their conserved modified amino acid sequences, and the amino acid sequences of the three complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region of Ab6 are shown in SEQ ID NO. 30-32 or their conserved modified amino acid sequences.

[0013] The CDR3 of the heavy chain variable region of Ab5 has the same amino acid sequence as the CDR3 of the heavy chain variable region of Ab6. The CDR3 of the light chain variable region of Ab5 has the same amino acid sequence as the CDR3 of the light chain variable region of Ab6.

[0014] Preferably, the antibody is a rabbit monoclonal or polyclonal antibody or a fragment thereof.

[0015] Preferably, the antibody is a humanized antibody or a chimeric antibody.

[0016] Preferably, the CDRs have 47.4%-100% homology.

[0017] Preferably, the amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of the monoclonal antibody against human sCD14-ST protein are as shown in SEQ ID NO.33-36, and the amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the light chain variable region of Ab1 are as shown in SEQ ID NO.37-40.

[0018] The amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of Ab2 are shown in SEQ ID NO.41-44, and the amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the light chain variable region of Ab2 are shown in SEQ ID NO.45-48.

[0019] The amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of Ab3 are shown in SEQ ID NO.49-52, and the amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the light chain variable region of Ab3 are shown in SEQ ID NO.53-56.

[0020] The amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of Ab4 are shown in SEQ ID NO.57-60, and the amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the light chain variable region of Ab4 are shown in SEQ ID NO.61-64.

[0021] The amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of Ab5 are shown in SEQ ID NO.65-68, and the amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the light chain variable region of Ab5 are shown in SEQ ID NO.69-72.

[0022] The amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of Ab6 are shown in SEQ ID NO.73-76, and the amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the light chain variable region of Ab6 are shown in SEQ ID NO.77-80.

[0023] Preferably, in the monoclonal antibody against human sCD14-ST protein, the amino acid sequence of the heavy chain of Ab1 is shown in SEQ ID NO.81, and the amino acid sequence of the light chain of Ab1 is shown in SEQ ID NO.82;

[0024] The amino acid sequence of the heavy chain of Ab2 is shown in SEQ ID NO.83, and the amino acid sequence of the light chain of Ab2 is shown in SEQ ID NO.84.

[0025] The amino acid sequence of the heavy chain of Ab3 is shown in SEQ ID NO.85, and the amino acid sequence of the light chain of Ab3 is shown in SEQ ID NO.86.

[0026] The amino acid sequence of the heavy chain of Ab4 is shown in SEQ ID NO. 87; the amino acid sequence of the light chain of Ab4 is shown in SEQ ID NO. 88.

[0027] The amino acid sequence of the heavy chain of Ab5 is shown in SEQ ID NO.89, and the amino acid sequence of the light chain of Ab5 is shown in SEQ ID NO.90.

[0028] The amino acid sequence of the heavy chain of Ab6 is shown in SEQ ID NO.91, and the amino acid sequence of the light chain of Ab6 is shown in SEQ ID NO.92.

[0029] In this application, "conservative modification" refers to amino acid modifications that do not substantially affect the properties of the original sequence antibody, including amino acid mutations, insertions, and deletions. Specific modification methods can be those known in the art, such as site-directed mutagenesis or PCR-mediated mutagenesis. Conservative substitution of amino acids refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. The amino acid residue family is defined in the art and includes amino acids with basic side chains (such as lysine, arginine, and histidine), acidic side-chain amino acids (such as aspartic acid and glutamic acid), uncharged polar side-chain amino acids (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side-chain amino acids (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side-chain amino acids (such as threonine, valine, and isoleucine), and aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine). Therefore, one or more amino acid residues in the CDR region can be replaced by other amino acid residues from the same side chain family, and detection is performed to ensure that the original sequence function is preserved.

[0030] "Conservatively modified variants" in this application include individual amino acids in a polypeptide sequence that are substituted, deleted, or added by functionally similar amino acids, as well as polymorphic variants, interspecific homologues, and their alleles. The following groups of amino acids can be substituted for each other: (1) alanine (A), glycine (G); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); (6) phenylalanine (F), tyrosine (Y), tryptophan (W); (7) serine (S), threonine (T); (8) cysteine ​​(C), methionine (M). "Conservative sequence modification" in this application refers to amino acid modifications that substantially do not affect or alter their binding affinity.

[0031] Preferably, the monoclonal antibody against human sCD14-ST protein further includes covalently or non-covalently linked conjugates.

[0032] Preferably, the FRs have 55.6%-100% homology.

[0033] More preferably, the monoclonal antibody against human sCD14-ST protein, wherein the conjugate includes an enzyme, a fluorescent protein, a fluorophore, biotin, or streptavidin.

[0034] More preferably, the enzyme includes HRP.

[0035] The present invention also provides the application of the above-mentioned monoclonal antibody against human sCD14-ST protein in the preparation of detection products for human sCD14-ST protein, wherein the detection products are test strips, test kits, etc.

[0036] A detection product for human sCD14-ST protein, comprising any of the above-mentioned anti-human sCD14-ST monoclonal antibodies.

[0037] Preferably, the testing product is a test strip or a test kit.

[0038] More preferably, the test strip is a colloidal gold test strip or an ELISA test strip; the detection kit is a colloidal gold detection kit, an ELISA detection kit, an immunoturbidimetric detection kit, an immunochromatographic kit, a magnetic particle detection kit, a chemiluminescence detection kit, an immunofluorescence detection kit, or a radioimmunoassay kit.

[0039] Compared with the prior art, the advantages of the present invention are as follows: The present invention targets the human sCD14-ST protein and has obtained a rabbit anti-human monoclonal antibody. This antibody or the corresponding detection kit has higher specificity, affinity and sensitivity, and can accurately quantify the human sCD14-ST protein; it can play an important role in diagnostic and prognostic detection, as well as in the study of the dynamic mechanism of human sCD14-ST protein in the field of immunology. Attached Figure Description

[0040] Figure 1 The image shows the results of affinity determination of monoclonal antibodies Ab1, Ab2, Ab3, Ab4, Ab5 and Ab6 using the ELISA method.

[0041] Figure 2 The graph shows the affinity determination results of monoclonal antibody Ab3 using the Biacore method.

[0042] Figure 3 The image shows the affinity determination results of monoclonal antibody Ab5 using the Biacore method. Detailed Implementation

[0043] In this application, "antibody" is a broad concept, encompassing various antibody structures, including but not limited to Y-shaped antibodies (i.e., full-length antibodies), antigen-binding portions of Y-shaped antibodies, and genetic or chemical modifications of both. The antigen-binding portion refers to one or more fragments of a Y-shaped antibody, wherein said one or more fragments retain the antibody's ability to specifically bind to the sCD14-ST protein. Prior art has demonstrated that the antigen-binding function of full-length antibodies can be achieved through their fragments.

[0044] In this application, "monoclonal antibody (mAb)" refers to a substantially homogeneous antibody, where each antibody is essentially identical, although a small number of naturally occurring mutations may exist in individual antibodies. Monoclonal antibodies exhibit specificity and affinity for specific antigenic epitopes. Unlike polyclonal antibodies, which include multiple different antibodies targeting different antigenic epitopes, monoclonal antibodies generally target only the same or substantially identical epitopes. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous group of antibodies, without requiring production through any specific method. In fact, antibodies can be prepared using various methods, such as single B cell culture and cloning, hybridoma, recombinant DNA, or phage antibody library isolation and screening.

[0045] In this application, "anti-human sCD14-ST protein mAb" refers to a monoclonal antibody capable of binding to the human sCD14-ST protein with sufficient affinity, thereby enabling its use in detection, diagnostic, and / or therapeutic agents targeting the human sCD14-ST protein. The term "affinity" in this application refers to the strength of the total non-covalent intermolecular interaction between a single binding site of a molecule (e.g., an antibody) and its binding chaperone (e.g., an antigen). Intermolecular interactions include hydrogen bonds, electrostatic interactions, hydrophobic forces, and van der Waals forces.

[0046] In this application, the modifier "rabbit" in terms such as "rabbit antibody" and "rabbit anti-human sCD14-ST protein mAb" indicates that the antibody's complementarity-determining region (CDR) originates from a rabbit germline immunoglobulin sequence. A rabbit antibody or rabbit anti-human sCD14-ST protein mAb comprises an antibody whose CDR sequence originates from a rabbit germline immunoglobulin sequence. Preferably, a rabbit antibody or rabbit anti-human sCD14-ST protein mAb comprises an antibody whose CDR sequence originates from a rabbit germline immunoglobulin sequence, while its antibody backbone region (FR) originates from an immunoglobulin sequence of another mammal (such as a mouse or human). Preferably, a rabbit antibody or rabbit anti-human sCD14-ST protein mAb comprises an antibody whose FR and CDR both originate from a rabbit germline immunoglobulin sequence. The "rabbit antibody" or "rabbit anti-human sCD14-ST protein mAb" in this application may also be an antibody that does not contain amino acid residues encoded by a rabbit germline immunoglobulin sequence, for example, an antibody induced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo. However, the “rabbit antibody” or “rabbit anti-human sCD14-ST protein mAb” in this application does not include antibodies whose CDR sequences are derived from other mammals (e.g., mice).

[0047] In the embodiments of this application, the rabbit anti-human sCD14-ST protein mAb is a Y-shaped antibody comprising a heavy chain and a light chain. The heavy chain includes a variable region (VH) and at least one constant region (CH). The VH is generally located at the N-terminus of the heavy chain and has higher variability in its amino acid sequence compared to the CH. The VH of different antibodies can be different and can specifically recognize each antibody. In isotypes or of the same type of antibody, the amino acid sequence of the CH can be the same, but the amino acid sequence of the CH is different for different types. In this application, "isotype" refers to the antibody class (e.g., IgG) encoded by the heavy chain constant region gene. Mammalian antibodies have five different types of heavy chains: γ, δ, α, μ, and ε, representing five types of antibodies: IgG, IgD, IgA, IgM, and IgE, respectively. Rabbits have at least four isotypes: IgA, IgE, IgG, and IgM; while humans and mice have the above five antibody isotypes.

[0048] The light chain is a small polypeptide subunit relative to the heavy chain, consisting of a variable region (VL) and a constant region (CL). The VL is generally located at the N-terminus of the light chain and has greater variability in its amino acid sequence compared to the CL. The VH can be different for different antibodies and is specific to the amino acid sequence of each antibody.

[0049] In embodiments of this application, the variable regions VH and VL are responsible for recognizing and binding to the human sCD14-ST protein. Preferably, CH and CL do not directly contact the residues of the human sCD14-ST protein.

[0050] Each variable region (VH) and VL includes a complementarity-determining region (CDR) and a backbone region (FR). The CDR is a hypervariable region within the variable region, containing antigen-contacting residues that enable the Y-shaped rabbit monoclonal antibody to recognize and contact the human sCD14-ST protein. The monoclonal antibody against human sCD14-ST protein provided in the embodiments of this application contains six CDRs, three in the VH region and three in the VL region.

[0051] Adjacent CDRs in the VH and VL regions are separated by FRs, meaning that monoclonal antibodies against human sCD14-ST protein contain 8 FRs, 4 of which are in the VH region and the other 4 are in the VL region. FRs are conserved regions of the variable region and can act as a scaffold to enable CDRs to have a three-dimensional structure that can contact antigens (such as the extracellular domain of human sCD14-ST protein).

[0052] The CDR region determines the specificity and affinity of monoclonal antibodies against sCD14-ST protein. The FR region helps maintain the overall structure of the variable region of the monoclonal antibody against sCD14-ST protein and supports the CDR so that it can bind to the antigen in a suitable conformation.

[0053] The three-dimensional structures of different antibody FRs can be identical. In some embodiments, the CDR of a monoclonal antibody can be transferred to the FR of another antibody from a different species while retaining its ability to bind to the sCD14-ST protein, to form a chimeric antibody. For example, the CDR of a rabbit anti-sCD14-ST protein monoclonal antibody can be transplanted into the FR of a human antibody to form a humanized antibody against the human sCD14-ST protein.

[0054] There are various methods for preparing rabbit anti-human sCD14-ST protein, including monoclonal antibody methods, such as cell hybridization, including but not limited to viral transformation or primary transformation of B lymphocytes, and it can also be obtained through single B cell preparation technology.

[0055] Immunization, isolation, and fusion of spleen cells can be performed using conventional methods in the field. For example, an immunogen containing the extracellular domain of the human sCD14-ST protein can be obtained by stimulating an immune response in New Zealand white rabbits. Specifically, this involves isolating sCD14-ST protein-specific B cells from PBMCs and secondary lymphoid tissues, and amplifying candidate IgG genes to recombinantly express rabbit mAbs. The binding affinity and specificity of the mAb candidates are verified by direct antigen ELISA, and capture ELISA and sandwich ELISA are used to further characterize mAbs that recognize the human sCD14-ST protein for subsequent analysis and development.

[0056] There are various methods for isolating and purifying rabbit mAbs. For example, they can be isolated from the culture supernatant of mammalian cells transfected with rabbit antibody genes, and then purified using protein A affinity chromatography. The purity and functional effect can be verified by SDS-PAGE and ELISA.

[0057] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The terms "comprising," "including," and "having" in this application are open-ended descriptions, meaning "including but not limited to." Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention.

[0058] Experimental Example 1: Screening, isolation, and purification of monoclonal antibodies against human sCD14-ST protein

[0059] The applicant used rabbit single B cell antibody development technology to isolate sCD14-ST specific single B cells from the peripheral blood of rabbits immunized with sCD14-ST, and obtained multiple antibodies that specifically recognize anti-sCD14-ST protein through PCR amplification and sequencing analysis.

[0060] 1. Animal immunization

[0061] To obtain a rabbit monoclonal antibody that recognizes human sCD14-ST protein, our company used our self-produced recombinant human sCD14-ST protein (amino acid sequence as shown in SEQ ID NO. 97) as an immunogen to immunize New Zealand white rabbits. Each rabbit was immunized with 250 μg. For the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant to form an emulsion, which was injected subcutaneously at multiple sites on the abdomen and back. Two weeks later, 250 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant to form an emulsion, which was injected subcutaneously at multiple sites on the abdomen and back. Three booster immunizations were performed. After the three immunizations, the serum titer was measured by ELISA. Rabbits with high serum titers were selected for a booster immunization with 250 μg of the immunogen injected subcutaneously at multiple sites. The spleen was harvested three days later.

[0062] 2. Isolation and screening of peripheral blood PBMCs

[0063] Aseptically collect peripheral anticoagulated blood from rabbits, separate PBMC immune cells using Ficoll density gradient centrifugation, wash cells twice with sterile PBS, count suspended cells, and PBMC cells can be stored in liquid nitrogen for long-term preservation.

[0064] 3. Flow cytometry sorting and culture of single B cells

[0065] (1) Resuspend PBMCs in PBS and adjust the cell concentration to 1×10⁻⁶. 7 B cells were stained with flow cytometry antibody at a ratio of 1:100, according to the volume of resuspended cells. Goat Anti-rabbit IgM FITC and sCD14-ST specific-APC were added at a ratio of 1:100. Note that blank control and single staining control should be set up. Incubate at room temperature in the dark for 30 min. Wash twice with PBS, centrifuge at 300g for 5 min at 4 degrees.

[0066] (2) Resuspend cells in PBS and filter them through a 100-mesh filter before processing. Five minutes before processing, add 2% PI for staining to remove dead cells. Use controls to adjust voltage and fluorescence compensation settings. Use FSC-A / SSC-A to select lymphocytes and use FSC-W / FSC-A to remove adherent cells. Select IgM-negative, antigen-positive, and PI-negative B cells.

[0067] (3) By flow cytometry, add one B cell to each well of a 96-well plate containing 100 μl of complete 1640 medium (containing 10% FBS, 1% penicillin-streptomycin, 1% glutamine, and 2 μg / mL human IL2), centrifuge the 96-well plate at 300g for 5 minutes, and incubate at 37°C and 5% CO2 for 7 days.

[0068] 4. Screening for positive clones using conventional ELISA method

[0069] (1) Plate coating: sCD14-ST antigen, 5 μg / ml, 100 μl / well, overnight at 4°C;

[0070] (2) Blocking: 3% BSA-PBS, 300 μl / well, 37℃, 1.5h;

[0071] (3) Washing: PBST, 300 μl / well, twice;

[0072] (4) Sample addition: Take 50 μl of culture supernatant from a 96-well plate, dilute it with 50 μl of PBS at a ratio of 1:1, add 100 μl / well, incubate at 37℃ for 1 h;

[0073] (5) Washing: PBST, 300 μl / well, 3 times;

[0074] (6) Add Goat-anti Rabbit IgG secondary antibody: 100 μl / well, 37℃, 30 min;

[0075] (7) Washing: PBST, 300 μl / well, 3 times;

[0076] (8) Add TMB Substrate Solution, 100 μl / well, 37℃, 5-10 min;

[0077] (9) Termination: 2M HCl, 50μl / well;

[0078] (10) Readings: OD450-OD630.

[0079] The test results are shown in Table 1 below.

[0080] Table 1. Results of ELISA screening for positive clones (data marked with "*" indicates strongly positive monoclonal antibodies).

[0081]

[0082]

[0083] 5. Sequencing and expression of specific rabbit monoclonal antibodies

[0084] B cells corresponding to ELISA-positive antibodies were collected, and RNA was extracted and reverse transcribed into cDNA using conventional methods.

[0085] The primer sequences for the heavy chain variable region gene are as follows:

[0086] Forward primer: 5'-aagcttgccaacatgcagactgggctgcgctggcttc-3', as shown in SEQ ID NO.93;

[0087] Reverse primer: 5'-ccattggtgagcatgcccgag-3', as shown in SEQ ID NO.94;

[0088] The primers for amplifying the light chain variable region gene are:

[0089] Forward primer: 5'-aagcttgctaccatgcacagagggcccccactc-3', as shown in SEQ ID NO.95;

[0090] Reverse primer: 5'-cagtgtctgctgaggatgtaggtac-3', as shown in SEQ ID NO.96;

[0091] The heavy and light chain variable region gene was amplified using cDNA as a template. The PCR reaction system was as follows: 95℃ for 3 min, (95℃ for 1 min, 58℃ for 30 s, 72℃ for 1 min) for a total of 25 cycles, and finally 72℃ for 5 min. The PCR product was then recovered and purified.

[0092] The heavy and light chain variable region gene of the rabbit recombinant monoclonal antibody was ligated into a T vector for transformation, plated, and finally verified by colony PCR. The gene sequence of the variable region gene of the rabbit monoclonal antibody was obtained by sequencing the gene of the positive clone.

[0093] The heavy chain variable region gene of the correctly sequenced rabbit monoclonal antibody was subcloned into the PATX1-Rabbit H plasmid, and the light chain variable region gene was subcloned into the PATX1-Rabbit L plasmid. The two plasmids were co-transfected into Xten CHO cells. After 72 hours of transfection, the cells were removed by centrifugation, and the culture supernatant was purified with Protein A resin.

[0094] The final selected monoclonal antibodies against human sCD14-ST protein are Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6. Among them:

[0095] The amino acid sequences of the three complementary determinant regions CDR1, CDR2 and CDR3 of the heavy chain variable region of Ab1 are shown in SEQ ID NO. 1-3, and the amino acid sequences of the three complementary determinant regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO. 4-6.

[0096] The amino acid sequences of the three complementary determinant regions CDR1 and CDR3 of the heavy chain variable region of Ab2 are shown in SEQ ID NO.7-8, and the amino acid sequence of CDR2 is STG; the amino acid sequences of the three complementary determinant regions CDR1, CDR2 and CDR3 of the light chain variable region of Ab2 are shown in SEQ ID NO.9-11.

[0097] The amino acid sequences of the three complementary determinant regions CDR1 and CDR3 of the heavy chain variable region of Ab3 are shown in SEQ ID NO.12-13, and the amino acid sequence of CDR2 is SSD; the amino acid sequences of the three complementary determinant regions CDR1, CDR2 and CDR3 of the light chain variable region of Ab3 are shown in SEQ ID NO.14-16.

[0098] The amino acid sequences of the three complementary determinants of the heavy chain variable region of Ab4, CDR1, are shown in SEQ ID NO.17; the amino acid sequence of CDR2 is DSG; and the amino acid sequence of CDR3 is GDL. The amino acid sequences of the three complementary determinants of the light chain variable region of Ab4, CDR1, CDR2, and CDR3, are shown in SEQ ID NO.18-20.

[0099] The amino acid sequences of the three complementary determinant regions CDR1, CDR2 and CDR3 of the heavy chain variable region of Ab5 are shown in SEQ ID NO.21-23, and the amino acid sequences of the three complementary determinant regions CDR1, CDR2 and CDR3 of the light chain variable region of Ab5 are shown in SEQ ID NO.24-26.

[0100] The amino acid sequences of the three complementarity-determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of Ab6 are shown in SEQ ID NO.27-29, and the amino acid sequences of the three complementarity-determining regions CDR1, CDR2 and CDR3 of the light chain variable region of Ab6 are shown in SEQ ID NO.30-32.

[0101] The amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of Ab1 are shown in SEQ ID NO.33-36, and the amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the light chain variable region of Ab1 are shown in SEQ ID NO.37-40.

[0102] The amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of Ab2 are shown in SEQ ID NO.41-44, and the amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the light chain variable region of Ab2 are shown in SEQ ID NO.45-48.

[0103] The amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of Ab3 are shown in SEQ ID NO.49-52, and the amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the light chain variable region of Ab3 are shown in SEQ ID NO.53-56.

[0104] The amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of Ab4 are shown in SEQ ID NO.57-60, and the amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the light chain variable region of Ab4 are shown in SEQ ID NO.61-64.

[0105] The amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of Ab5 are shown in SEQ ID NO.65-68, and the amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the light chain variable region of Ab5 are shown in SEQ ID NO.69-72.

[0106] The amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of Ab6 are shown in SEQ ID NO.73-76, and the amino acid sequences of the four backbone regions FR1, FR2, FR3 and FR4 of the light chain variable region of Ab6 are shown in SEQ ID NO.77-80.

[0107] Accordingly, the amino acid sequence of the heavy chain of Ab1 is shown in SEQ ID NO.81, and the amino acid sequence of the light chain of Ab1 is shown in SEQ ID NO.82;

[0108] The amino acid sequence of the heavy chain of Ab2 is shown in SEQ ID NO.83, and the amino acid sequence of the light chain of Ab2 is shown in SEQ ID NO.84.

[0109] The amino acid sequence of the heavy chain of Ab3 is shown in SEQ ID NO.85, and the amino acid sequence of the light chain of Ab3 is shown in SEQ ID NO.86.

[0110] The amino acid sequence of the heavy chain of Ab4 is shown in SEQ ID NO. 87; the amino acid sequence of the light chain of Ab4 is shown in SEQ ID NO. 88.

[0111] The amino acid sequence of the heavy chain of Ab5 is shown in SEQ ID NO.89, and the amino acid sequence of the light chain of Ab5 is shown in SEQ ID NO.90.

[0112] The amino acid sequence of the heavy chain of Ab6 is shown in SEQ ID NO.91, and the amino acid sequence of the light chain of Ab6 is shown in SEQ ID NO.92.

[0113] Experimental Example 2: Affinity Identification of Recombinant Expression Antibodies

[0114] The recombinant antibody was analyzed for affinity using both ELISA and Biacore methods. The Biacore reagent kit information provided is: CM5 sensor chip, GE 10305570.

[0115] Human Antibody Capture Kit, GE BR-1008-39, lot 10302676;

[0116] EDC / NHS Kit GE BR-1000-50 / lot 30649;

[0117] Running buffer HBS-EP+: 10mM Hepes (sigma, H3375), 3mM EDTA (10009717, 150mM), NaCl2 (Sinopharm, 10019318), 0.05% T (Beyotime, ST825);

[0118] 1. Identification of the affinity of recombinant expressed antibodies using ELISA method

[0119] The method is the same as step 4 of Experimental Example 1. The antibody detection range is: 0.0006553 μg / ml, 0.001638 μg / ml, 0.004096 μg / ml, 0.01024 μg / ml, 0.0256 μg / ml, 0.064 μg / ml, 0.16 μg / ml, 0.40 μg / ml, 1.00 μg / ml, 2.50 μg / ml, 6.25 μg / ml, and 15.625 μg / ml.

[0120] The affinity determination results of the Elisa method are as follows: Figure 1 As shown in Table 1 below.

[0121] Table 2. Affinity determination results of recombinant expressed antibodies by ELISA.

[0122]

[0123]

[0124] from Figure 1 As shown in Table 2, the three antibodies with the best affinity were Ab5 (EC50 6.23 ng / ml), Ab3 (EC50 8.44 ng / ml), and Ab2 (EC50 11.03 ng / ml).

[0125] 2. Biacore method for identifying the affinity of recombinant expressed antibodies

[0126] The specific method is as follows:

[0127] (1) Power on the Biacore T200 instrument according to the standard operating procedure;

[0128] (2) Open the Biacore T200 control software and install the CM5 chip according to the standard procedure;

[0129] (3) Prepare to start the formal experiment. Start the operation. The buffer solution will flush the entire internal flow path system at a high flow rate. Standby.

[0130] (4) Select the appropriate test tube module based on the sample size;

[0131] (5) Begin capturing the chip; the coupling buffer is HEPES-ET. Prepare sufficient sample volume, EDC / NHS, and blocking buffer according to the sample rack position table. Place the sCD14-ST in the corresponding position on the test tube rack as required by the software. Close the test tube rack lid and return the sample rack to the sample chamber. The system will then automatically run the coupling procedure.

[0132] (6) After the coupling is completed, the sample detection is started. According to the standard procedure, the ligand sCD14-ST is set with a contact time of 30s and a flow rate of 10μl / min. The analyte Ab is set with a contact time of 120s, a flow rate of 30μl / min, a dissociation time of 300s, and the regeneration conditions are 3M magnesium chloride and a regeneration time of 30s.

[0133] (7) Prepare the corresponding samples to be tested as required, place them in the sample chamber according to the location requirements, and start the automatic running program for testing;

[0134] (8) Results analysis: Based on the results, perform data fitting analysis to obtain the final affinity fitting KD value.

[0135] Test results are as follows Figure 2 , 3 As shown in Table 2, the optimal affinity of the three antibodies obtained through curve fitting and calculation is 1.43E-13M for Ab3, 1.86E-13M for Ab5, and 8.96E-13M for Ab4.

[0136] Table 1. Affinity determination results of recombinant expressed antibodies using the Biacore method.

[0137] Ligand Analyte Affinity (KD) 1 sCD14-ST Ab3 5.19E-11 2 sCD14-ST Ab5 1.19E-10

[0138] Calculations show that the affinity of the rabbit monoclonal antibody provided by this invention is 10-100 times that of rodent monoclonal antibodies.

[0139] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody against human sCD14-ST protein, characterized in that, For monoclonal antibody Ab3; the amino acid sequences of 3 complementarity determining regions CDR1 and CDR3 of the heavy chain variable region of the monoclonal antibody Ab3 are shown as SEQ ID NO. 12-13, and the amino acid sequence of CDR2 is SSD; the amino acid sequences of 3 complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region of Ab3 are shown as SEQ ID NO. 14-16.

2. The monoclonal antibody against human sCD14-ST protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain of the monoclonal antibody Ab3 is shown as SEQ ID NO. 85, and the amino acid sequence of the light chain of Ab3 is shown as SEQ ID NO.

86.

3. The monoclonal antibody against human sCD14-ST protein according to claim 1, characterized in that, Also included are conjugates covalently or non-covalently linked, selected from enzymes, fluorescent proteins, fluorophores, biotin or streptavidin.

4. Use of the monoclonal antibody against human sCD14-ST protein according to any one of claims 1-3 in the preparation of a detection product for human sCD14-ST protein.

5. A detection product of human sCD14-ST protein, characterized by, The monoclonal antibody against human sCD14-ST according to any one of claims 1-3 is included.

6. The detection product of human sCD14-ST protein according to claim 5, characterized by, The detection product is a test strip or a test kit.

7. The detection product of human sCD14-ST protein according to claim 6, characterized by, The test strip is a colloidal gold test strip or an ELISA test strip; the test kit is a colloidal gold test kit, an ELISA test kit, an immunoturbidimetry test kit, an immunochromatography test kit, a magnetic particle test kit, a chemiluminescence test kit, an immunofluorescence test kit or a radioimmunoassay test kit.

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