Preparation method and application of rT3 recombinant monoclonal antibody

Abstract

The present invention relates to the field of antibody technology, and specifically to a method for preparing and applying a recombinant monoclonal antibody against rT3. The present invention utilizes phage display technology to screen and obtain the rT3 recombinant rabbit monoclonal antibody of the present invention, wherein the heavy chain variable region thereof has the amino acid sequence shown in SEQ ID NO:1; and the light chain variable region thereof has the amino acid sequence shown in SEQ ID NO:2. The monoclonal antibody has strong specificity and high detection sensitivity, and solves the problem of low rT3 content in samples and high detection difficulty. It has been identified that repeated multiple detections using the monoclonal antibody have good stability and high accuracy. The antibody is used for the detection of rT3 content in serum, and combined with other thyroid index tests and clinical manifestations, it can diagnose hyperthyroidism and hypothyroidism, and is also clinically significant in the detection of diabetes, lung infection, myocardial infarction, cirrhosis, uremia, and some cancers.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to a method for preparing rT3 recombinant monoclonal antibody and its application. Background Art

[0002] Reverse triiodothyronine (rT3) is a metabolically inactive thyroid hormone found in normal human serum. 95% of circulating rT3 is produced by the removal of an iodine residue from the tyrosine ring of T4 by 5-deiodinase in tissues such as the liver, kidney, pituitary gland, and myocardium. Approximately 99% of rT3 in serum is bound to the major serum binding proteins: thyroid-binding globulin (TBG), transthyretin (TTE), and albumin (Alb). Therefore, the level of serum binding proteins significantly influences rT3 measurement results. The metabolism of thyroxine in the body is reflected in the ratio of T4, T3, and rT3 in serum. However, rT3 is degraded more rapidly in the body, approximately three times faster than T3, with a half-life of 30 to 60 minutes.

[0003] The normal human body's rT3 content ranges from approximately 90 to 350 pg / mL. The concentration of rT3 in normal human serum is very low, only one-fourth to one-fifth of the concentration of T3. The daily production rate of rT3 in normal individuals is roughly equal to that of T3. In cases of hyperthyroidism or hypothyroidism, the trend of serum rT3 concentration changes mirrors that of T3 and T4. In other diseases, serum rT3 concentrations increase while serum T3 concentrations decrease.

[0004] In some thyroid-related diseases, such as Graves' hyperthyroidism, the elevated rT3 content in serum is basically consistent with the levels of T3 and T4. After treatment with antithyroid drugs and the beta-blocker propranolol, the concentration of rT3 decreases simultaneously with the concentrations of T3 and T4, but the rate of decrease is slower than that of T3 and T4. The concentrations are: T3>T4>rT3. The reason for this is that beta-blockers inhibit the activity of 5'-deiodinase, affecting the metabolic pathway, resulting in a decrease in the conversion of T4 to T3 and an increase in the conversion to rT3, and antithyroid drugs affect the clearance rate of rT3.

[0005] In studies of certain mild hyperthyroidism and hypothyroidism, such as subclinical hypothyroidism and chronic lymphocytic thyroiditis, rT3 changes earlier than T3 and T4. Although rT3 is synthesized in very small amounts in the thyroid gland and its diagnostic value is inferior to that of T3 and T4, subtle changes in hyperthyroidism can be promptly reflected in rT3 levels through its effects on 5-deiodinase activity in metabolic pathways. Therefore, serum rT3 measurement can aid in the diagnosis of hyperthyroidism and hypothyroidism, particularly in mild cases, where rT3 changes may be more sensitive than T3 and T4.

[0006] During thyroid disease treatment, patients whose hypothyroidism improves will typically have their T3 levels essentially return to normal, but the rT3 / T3 ratio will be lower than normal. This is because rT3 returns to normal more slowly. Therefore, a comprehensive assessment of thyroid function requires a combined analysis of T3, T4, TSH, and other test values. Simultaneous elevations in T3 and rT3 suggest a medication overdose. Following treatment with antithyroid drugs, as hyperthyroidism symptoms subside, serum rT3 levels decrease along with the decline in T3 and T4. This decrease in rT3 is slower than that of T3 and T4, and a decrease in rT3 suggests a medication overdose. Therefore, serum rT3 measurement can also be used to monitor medication use during treatment.

[0007] During pregnancy, elevated rT3 levels in amniotic fluid and significantly higher rT3 levels in the umbilical cord blood of newborns compared to adults, but with slightly lower T3 levels, can be used as a diagnosis of congenital hypothyroidism. Elevated rT3 concentrations are easier to measure, so rT3 measurements can more accurately reflect the thyroid function of the fetus and newborn.

[0008] rT3 levels are low in serum, so improving detection sensitivity requires high standards for immunoassay methods and antibody specificity. Common assays include radioimmunoassay, fluorescence immunoassay, enzyme-linked immunosorbent assay, luminescence immunoassay, electroluminescence immunoassay, and chemiluminescence immunoassay. Chemiluminescence immunoassay offers advantages such as high sensitivity, simplicity and speed, stable results, minimal error, safety, and long-term use. Therefore, monoclonal antibodies are used in chemiluminescence immunoassay reagents.

[0009] Phage display technology is a unique gene recombinant expression technique and a simple, effective screening tool. Its basic principle is to insert a foreign gene into the appropriate position of a phage structural gene through genetic engineering. When the phage infects Escherichia coli, the foreign gene and the structural gene are fused and expressed together to form a fusion protein. The displayed peptide or protein has excellent biological activity and can maintain the original physiological function and viability of the phage. It is then biopanned to remove free phage that have not bound to the target and screen for phage that specifically bind to the target molecule. These specifically bound phage are then eluted with acid and propagated by infecting host cells. This "adsorption-elution-amplification" process typically requires three to five cycles to highly enrich phage that specifically bind to the target molecule. The selected phage that specifically binds to the target molecule undergoes sequencing and bioinformatics analysis to obtain information about the target peptide, which is then artificially synthesized for further research. Using phage display technology to improve the performance of rT3 antibodies and solve the problem of rT3 detection is an urgent problem to be solved in this field. Summary of the Invention

[0010] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing rT3 recombinant monoclonal antibody and its application.

[0011] The present invention provides an rT3 monoclonal antibody, which is obtained by immunizing New Zealand white rabbits with rT3-6C-KLH antigen prepared by coupling rT3-6C with KLH and then screening using phage display technology. Compared with other antibodies, the rT3 monoclonal antibody has high affinity and strong specificity.

[0012] In the present invention, the rT3 monoclonal antibody,

[0013] The amino acid sequence of the CDR region of its heavy chain comprises at least one of the sequences shown in SEQ ID NO: 5, 6 or 7;

[0014] The amino acid sequence of the CDR region of the light chain thereof comprises at least one of the sequences shown in SEQ ID NO: 8, DAS or SEQ ID NO: 9.

[0015] Furthermore, the rT3 monoclonal antibody of the present invention,

[0016] The amino acid sequences of the CDR region of its heavy chain are GFSLDDYT, IYDNGRT, and ARKGAFGPDNAINSL;

[0017] The amino acid sequences of the CDR region of its light chain are QSVYNNNQ, DAS and LGRYSSGDRRA.

[0018] Furthermore, the heavy chain variable region of the rT3 monoclonal antibody has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the rT3 monoclonal antibody has the amino acid sequence shown in SEQ ID NO: 2.

[0019] In the present invention, the rT3 monoclonal antibody further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is of rabbit IgG1 subtype, and the light chain constant region is of rabbit K1 type.

[0020] The present invention provides a nucleic acid encoding the rT3 monoclonal antibody.

[0021] Furthermore, the nucleic acid encoding the rT3 monoclonal antibody comprises:

[0022] A nucleic acid encoding the heavy chain variable region of the rT3 monoclonal antibody, having a nucleotide sequence as shown in SEQ ID NO: 3;

[0023] The nucleic acid encoding the light chain variable region of the rT3 monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 4.

[0024] Furthermore, the nucleic acid encoding the heavy chain variable region of the progesterone monoclonal antibody further comprises any one or more of the following a) to c):

[0025] a) having at least 80% homology to the nucleic acid shown in SEQ ID NO: 3 and encoding a protein having the same or similar function as the nucleic acid shown in SEQ ID NO: 3;

[0026] b) A nucleic acid in which one or more bases are modified, substituted, deleted or added as shown in SEQ ID NO: 3;

[0027] c) a nucleic acid that is complementary or partially complementary to the nucleic acid shown in SEQ ID NO: 3.

[0028] Furthermore, the nucleic acid encoding the light chain variable region of the progesterone monoclonal antibody further comprises any one or more of the following A) to C):

[0029] A), having at least 80% homology to the nucleic acid shown in SEQ ID NO: 4 and encoding a protein with the same or similar function as the nucleic acid shown in SEQ ID NO: 4;

[0030] B) a nucleic acid as shown in SEQ ID NO: 4 in which one or more bases are modified, substituted, deleted or added;

[0031] C), a nucleic acid that is complementary or partially complementary to the nucleic acid shown in SEQ ID NO: 4.

[0032] The present invention provides an expression module comprising a promoter, a terminator and the nucleic acid of the present invention.

[0033] The present invention also provides a transcription unit, which refers to a DNA sequence starting from a promoter and ending at a terminator. The promoter and terminator may also be flanked or interposed between regulatory segments, which may include a promoter, enhancer, transcription termination signal, polyadenylation sequence, replication origin, nucleic acid restriction site, transmembrane signal peptide, and homologous recombination site operably linked to the nucleic acid sequence, such as a promoter enhancer, ITR sequence, polyA, or MIS signal peptide.

[0034] The present invention provides a recombinant vector comprising a vector backbone and the nucleic acid of the present invention.

[0035] Furthermore, the sources of the vector backbone described in the present invention include plants, animals, bacteria, fungi, phages, or viruses, which are not limited by the present invention. The viral vectors include: adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, or lentiviral vectors, etc. The phage vectors include phagemids and helper vectors, and the phagemids include but are not limited to pBluescript II-KS (+), pcomb3XSS, pCANTAB5E or pKK233.3. The animals include mammals and non-mammals, and the mammalian expression vectors include but are not limited to pcDNA 3.1, pIRES, pTT3, pCEP4, pATX1, or pCHO1.0. The bacterial vectors include but are not limited to pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, pColdII, etc. The fungal vectors include but are not limited to pYES2, pYES3, pYES6, pAUR23, etc.

[0036] In some embodiments of the present invention, a phage single-chain antibody library was constructed using the phagemid vector pcomb3XSS to carry the nucleic acid of the rT3 monoclonal antibody and nucleic acids of other types of antibodies for screening of single-chain antibodies. The results showed that the phage containing the rT3 monoclonal antibody of the present invention had high affinity and strong specificity.

[0037] In other embodiments, the present invention uses the mammalian cell vector pCHO 1.0 as a backbone to integrate the nucleic acid encoding the rT3 monoclonal antibody to construct a mammalian cell expression vector for the rT3 monoclonal antibody. The expression vector is transfected or transformed into FD-CHOS cells and multiple rounds of screening are performed to obtain a stable cell line that highly expresses the rT3 monoclonal antibody. The purified antibody has high purity and concentration, meeting production requirements.

[0038] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences or elements necessary for expression of the operably linked coding gene in a specific host organism. The nucleic acid sequences or elements necessary for expression in viral, microbial, or mammalian cells include promoters, ribosome binding sites, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, the terms "plasmid" and "vector" are sometimes used interchangeably, as plasmids are the most commonly used vector form. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions and are known or become known in the art, including but not limited to plasmids, phage particles, viral vectors, and / or simply potential genomic inserts.

[0039] The present invention provides a host cell transformed or transfected with the recombinant vector of the present invention.

[0040] Furthermore, the host cell described in the present invention also includes a host cell formed by co-transformation or transfection of the recombinant vector described in the present invention and its helper plasmid, and the present invention is not limited to this.

[0041] Furthermore, the transformation methods include chemical transformation and electroporation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection methods include adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0042] The host cells provided by the present invention may be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, but are not limited thereto. The present invention uses vectors constructed using recombinant DNA technology to transform or transfect host cells, so that the transformed host cells are capable of replicating protein-encoding vectors or expressing desired proteins.

[0043] Furthermore, the host cells of the present invention include mammalian cells, bacteria, or fungi. The mammalian cells include, but are not limited to, at least one of HEK-293, HEK293T, Hep G2, HELA, CHO-K1, COS-1, COS-7, NIH3T3, A204, A549, D-407, CHO, HCS-2, HT-29, U87, Sf9, or FD-CHOS. The bacteria include, but are not limited to, Escherichia coli. The fungi include, but are not limited to, yeast, which store the nucleic acid of the rT3 monoclonal antibody and / or are used for expression of the rT3 monoclonal antibody.

[0044] In some specific embodiments, the present invention uses mammalian FD-CHOS cells as recipient cells for the expression of the antibody.

[0045] The present invention provides a labeled antibody, which comprises a label and the rT3 monoclonal antibody of the present invention; the label comprises a chemical label or a biological label.

[0046] Furthermore, the chemical label is an isotope and / or a chemical drug.

[0047] Furthermore, the biomarker includes biotin, avidin or an enzyme label, and the enzyme label is preferably horseradish peroxidase or alkaline phosphatase.

[0048] The present invention provides a conjugate, which comprises a coupling medium and the rT3 monoclonal antibody of the present invention; the coupling medium is a solid medium or a semi-solid medium.

[0049] Furthermore, the coupling medium is selected from colloidal gold, polystyrene plates or beads.

[0050] The present invention provides a method for preparing rT3 monoclonal antibody, which comprises culturing the host of the present invention to obtain rT3 monoclonal antibody.

[0051] Use of any one of the following I) to VI) in the preparation of a product for detecting rT3 levels in serum:

[0052] 1), the rT3 monoclonal antibody of the present invention;

[0053] II), the nucleic acid of the present invention;

[0054] III), the expression vector of the present invention;

[0055] IV), the host cell of the present invention;

[0056] V), the labeled antibody of the present invention;

[0057] VI), the conjugate of the present invention;

[0058] VII), a culture containing rT3 monoclonal antibody prepared by the preparation method of the present invention.

[0059] In some specific embodiments, the rT3 recombinant rabbit monoclonal antibody provided by the present invention can be used as a detection antibody for the detection of anti-triiodothyronine with good sensitivity.

[0060] The rT3 recombinant monoclonal antibody provided by the present invention is suitable for immunological detection of anti-triiodothyronine.

[0061] The present invention provides a product for detecting rT3 in serum, which comprises any one or more of the following i) to iv):

[0062] i) the rT3 monoclonal antibody of the present invention;

[0063] ii), the labeled antibody of the present invention;

[0064] iii), the conjugate of the present invention;

[0065] iv) A culture containing rT3 monoclonal antibody prepared by the preparation method of the present invention.

[0066] Furthermore, the product of the present invention may be a test strip coated with the rT3 monoclonal antibody of the present invention.

[0067] Furthermore, the product of the present invention may be a kit, which further comprises: a coating buffer, a washing solution, a blocking solution, and / or a color developing solution.

[0068] In some embodiments, the kit is suitable for magnetic microparticle chemiluminescence detection of rT3. The results show that repeated detection using the monoclonal antibody has good stability and high accuracy.

[0069] The present invention also provides a method for detecting rT3, which comprises using the product of the present invention to perform rT3 detection in a blood sample.

[0070] The present invention utilizes phage display technology to screen and obtain the rT3 recombinant rabbit monoclonal antibody described herein. Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO:1, and its light chain variable region has the amino acid sequence shown in SEQ ID NO:2. This monoclonal antibody has strong specificity and high detection sensitivity, addressing the challenges of low rT3 levels in samples and the difficulty of detecting it. Repeated detection using this monoclonal antibody has demonstrated excellent stability and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Agarose gel electrophoresis diagram of extracted spleen RNA;

[0072] Figure 2 Agarose gel electrophoresis of the VL gene PCR product;

[0073] Figure 3 Agarose gel electrophoresis of VH gene PCR products;

[0074] Figure 4 Agarose gel electrophoresis diagram of scFv gene PCR product;

[0075] Figure 5Agarose gel electrophoresis diagram of the antibody library recombination rate bacterial liquid PCR identification;

[0076] Figure 6 This is the SDS-PAGE electrophoresis diagram of the recombinant antibody purification;

[0077] Figure 7 Correlation analysis between the antibodies of the present invention and antibodies from reference manufacturers;

[0078] Figure 8 It is the deviation between the test result and the theoretical concentration. DETAILED DESCRIPTION

[0079] The present invention provides rT3 recombinant rabbit monoclonal antibodies and their applications. Those skilled in the art can refer to the disclosure herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications herein to implement and apply the technology of the present invention without departing from the disclosure, spirit, and scope of the present invention.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by one of ordinary skill in the art. For definitions and terminology in this field, practitioners are referred to Current Protocols in Molecular Biology (Ausubel). Amino acid residue abbreviations are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0081] An antigen is a substance that can stimulate a specific immune response and bind to immune response products (antibodies and sensitized lymphocytes) both in vivo and in vitro, producing an immune effect (specific reaction). It is categorized as either complete antigens or haptens. Haptens, which are small molecules, are non-immunogenic and do not elicit an immune response. However, when haptens bind to larger proteins, they acquire immunogenicity and become complete antigens, stimulating the immune system to produce antibodies and effector cells.

[0082] Antibodies are immunoglobulins produced by plasma cells transformed from B lymphocytes in response to antigenic stimulation in animals. These immunoglobulins are capable of specifically binding to the corresponding antigen. The development of antibody production methods has progressed through three stages: In the first stage, antigens with multiple antigenic determinants were selected as immunogens and used to immunize animals using conventional methods. This activated the lymphocytes in the body, leading to the production of antibodies against multiple antigenic determinants. The resulting antiserum is known as polyclonal antibodies. In the second stage, hybridoma cells were formed by fusing hybridoma cells with B cells producing specific antibodies, resulting in the production of the first monoclonal antibody. In the third stage, with the rapid development of recombinant DNA technology and other molecular biology techniques, molecular biology methods were used to produce genetically engineered antibodies, known as third-generation antibodies. Compared to hybridoma monoclonal antibodies, third-generation antibodies can enrich for antigen-specific antibodies in an immune phage antibody library, producing more and better antibodies, even with higher affinity than antibodies obtained using traditional methods.

[0083] Most phage display technologies commonly use filamentous phages as vectors, such as M13, fd, and f1. Filamentous phages have five main capsid proteins: PIII, PVI, PVII, PVIII, and PIX. Fusion proteins used for antibody display primarily come in two types: PIII and PVIII. PIII is the largest capsid protein, and its spatial structure is flexible and mutable. The C-terminus of PIII is a hydrophobic region that anchors the capsid, while the N-terminus is free, allowing for the insertion of large foreign proteins without affecting the structure and function of the phage. The C-terminus of the PVIII protein primarily contains basic amino acid residues, making it easy to bind to DNA. The central region is a hydrophobic region that forms the phage capsid, while the N-terminus is free and exposed on the phage surface. Foreign sequences of 5-6 amino acid residues are typically fused to the N-terminus, with approximately 2700 copies per virion. Fusion proteins containing PVIII can be displayed polyvalently, and then affinity screening can be used to identify target genes. In the PⅧ protein display system, the copy number of the fusion protein is relatively high, generally around 2700, and generally accommodates antibody peptides of no more than 6 amino acids, otherwise it will affect phage assembly and reduce its infectivity. Because larger peptides or even entire protein gene fragments can be inserted into gPⅢ, fragments larger than 50 ku have been successfully displayed. In addition, the copy number of PⅢ is relatively low (around 5 copies), which is conducive to the screening of high-affinity ligands.

[0084] The general process for preparing a natural antibody phage library is to extract total RNA from immune cells (pre-, mature, and memory B cells), typically obtained from immune organs (bone marrow, spleen, lymph nodes, and tonsils) and peripheral blood. Reverse transcription is performed using 6-nucleotide random primers, poly T, or designed PCR downstream primers to generate first-strand cDNA. Upstream primers are designed based on the conserved 5' end of the antibody framework region FR1 or the guide sequence; downstream primers for ScFv libraries are designed from conserved sequences in the hinge region (J region), and for Fab antibody libraries from constant sequences. A drawback of screening is that with multiple rounds of phage panning, rare antibodies and those with lower affinity are often lost, resulting in lower affinity antibodies. However, the greater the diversity of the phage library, the higher the success rate of screening for high-affinity antibodies.

[0085] Antibodies exist as one or more Y-shaped monomers, each composed of four polypeptide chains: two identical heavy chains and two identical light chains. The variable region, located at the top of the Y-shaped structure, is the antigen-binding domain. The variable region of an antibody heavy or light chain is the N-terminal mature region of the chain. Human antibody heavy chains include α, δ, ε, γ, and μ, and the corresponding antibodies are called IgA, IgD, IgE, IgG, and IgM. Human γ chains can be further subdivided into γ1, γ2, γ3, and γ4 subclasses, corresponding to the IgG1, IgG2, IgG3, and IgG4 subtypes, respectively. Light chains come in two types: λ and κ. IgG1 is the most abundant subtype in plasma.

[0086] "Antibodies" include antibodies or immunoglobulins of any isotype, or antibody fragments that retain specific antigen binding, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. Antibodies can be labeled and detected, for example, using radioisotopes, enzymes that produce detectable substances, fluorescent proteins, biotin, and the like. Antibodies can also be bound to solid supports, including but not limited to polystyrene plates or beads, and the like.

[0087] The amino acid sequence encoding the heavy chain variable region of the rT3 monoclonal antibody is: QSLGESRGGLVTPGTPLTLTCTVSGFSLDDYTMTWVRQAPGKGLQWVGLIYDNGRTYYPSWAKGRFTISKTSTTVDLKMTSLTAADTATYFCARKGAFGPDNAINSLWGQGTLVTVSS (SEQ ID NO: 1).

[0088] The amino acid sequence encoding the light chain variable region of the rT3 monoclonal antibody is: AVVLTQTASPVSAALGSTVTINCQSSQSVYNNNQLSWYQQKPGQAPKRLIYDASKVSSGVPSRFRGSGSGTQFTLTISDVQCDDAATYYCLGRYSSGDRRAFGGGTKLEIK (SEQ ID NO: 2).

[0089] The nucleotide sequence encoding the heavy chain variable region of the rT3 monoclonal antibody is: cagtcgctgggggagtccaggggaggcctggtcacgcctgggacacccctgacactcacctgcacagtctctggattctccctcgatgattatacaatgacctgggtccgccaggctccagggaaggggctgcagtgggtcggcctcatttatgataatggtcgcacatattacccaagctgggcgaagggccggttcaccatctccaaaacctcgaccacggtggatctgaaaatgaccagtctgacagccgcggacacggccacctatttctgcgccagaaagggtgcttttggtcctgataatgctatcaatagtttgtggggccaaggcaccctggtcaccgtctcttca (SEQ ID NO: 3).

[0090] The nucleotide sequence encoding the light chain variable region of the rT3 monoclonal antibody is: gccgtcgtgctgacccagactgcatcccccgtgtctgcagctctgggaagcacagtcaccatcaattgccagtccagtcagagtgtttacaataataaccaattatcctggtatcagcagaaaccagggcaggctcctaaacgcctgatctatgatgcatccaaagtgtcatctggggtcccatcgcggttccgtggcagtggatctgggacacagttcactctcaccatcagcgacgtgcagtgtgacgatgctgccacttactactgtttgggccgttatagtagtggtgatcgtagagctttcggcggagggaccaaactggaaatcaaa (SEQ ID NO: 4).

[0091] The amino acid sequence of CDR1 in the heavy chain variable region is: GFSLDDYT (SEQ ID NO: 5).

[0092] The amino acid sequence of CDR2 in the heavy chain variable region is: IYDNGRT (SEQ ID NO: 6).

[0093] The amino acid sequence of CDR3 of the heavy chain variable region is: ARKGAFGPDNAINSL (SEQ ID NO: 7).

[0094] The amino acid sequence of CDR1 of the light chain variable region is: QSVYNNNQ (SEQ ID NO: 8).

[0095] The amino acid sequence of CDR2 of the light chain variable region is: DAS.

[0096] The amino acid sequence of CDR3 of the light chain variable region is: LGRYSSGDRRA (SEQ ID NO: 9).

[0097] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0098] The present invention will be further described below in conjunction with the embodiments:

[0099] Example 1 Preparation for phage screening platform provided by the present invention:

[0100] 1. Immunogen preparation:

[0101] A DMSO solution of rT3-6C (5.417 mg, 1.08 ml) was slowly added dropwise to a 0.01 M PBS solution of hemocyanin (KLH) (30 mg, 3 ml). The reaction was shaken at room temperature overnight. After completion of the reaction, the reaction solution was dialyzed five times against 0.01 M PBS (changing the solution every 2 hours, with the final dialysis performed overnight). This yielded the immunogen.

[0102] 2. Immunization of animals with recombinant immunogens:

[0103] New Zealand white rabbits were immunized five times with the prepared antigen rT3-6C-KLH. The immunization cycle lasted 30 days, with multiple subcutaneous injections at the back. The first immunization was performed with 2 mg of the immunizing antigen emulsified with an equal volume of Freund's complete adjuvant. Subsequently, 1 mg of the immunizing antigen was emulsified with an equal volume of Freund's incomplete adjuvant for five immunizations, with 30-day intervals between each immunization. Blood was collected from the ear vein on the 10th day after the fifth immunization. The blood was incubated at 37°C for 1 hour and then centrifuged at 6000 rpm for 10 minutes. The supernatant (antiserum) was collected and analyzed for ELISA.

[0104] 3. Antiserum titer detection

[0105] Prepare the test plate, add 0.05mol / L CB (pH9.6) coating buffer to the anti-rabbit antibody at a coating concentration of 4μg / mL, and coat overnight at 4℃. Test serum (431#, 432#, 433# correspond to the rabbit numbers of the blood samples taken) is diluted from 1 / 500, 50μl / well, and the benchmark manufacturer's antibody is set as a positive control. Incubate at 37℃ for 30min; wash 5 times with PBST, pat dry, add 1 / 2W diluted rT3 enzyme conjugate (horseradish peroxidase labeled antigen), 50μl / well, incubate at 37℃ for 30min; wash 5 times with PBST, pat dry, add luminescent substrate A and B solution, 50μl / well each, react in the dark for 5min, and measure the signal value. The test results are shown in Table 1. The titer reaches 10 5 After the final boost immunization, the animals were sacrificed three days later, spleen cells were extracted, and total RNA from spleen tissue was extracted by Trizol method (eg Figure 1 cDNA was synthesized by reverse transcription.

[0106]

[0107] Example 2 scFv gene splicing and phage screening construction

[0108] 1. scFv gene splicing:

[0109] PCR was used to amplify the light chain variable region and heavy chain variable region of the antibody, respectively. The PCR reaction procedure was as follows:

[0110]

[0111] The PCR products were recovered from 1% agarose gel. Figure 2 and Figure 3 The amplified light chain variable region and heavy chain variable region were spliced into scFv using overlap-PCR method, and the product was recovered through 1% agarose gel and stored at -20℃. Figure 4 shown.

[0112] 2. Construction and screening of phage single-chain antibody library:

[0113] The phagemid vector pcomb3XSS and the purified ScFv fragment were digested with SfiI to construct a recombinant plasmid. The recombinant plasmid was electroporated into TG1 competent cells to construct a rabbit-derived immune single-chain antibody library (the antibody library recombinant rate was determined by bacterial liquid PCR). Figure 5), and prepare a primary phage single-chain antibody library; the primary phage single-chain antibody library is enriched and screened for three rounds to obtain a specific phage single-chain antibody library with high affinity and strong specificity; a monoclonal clone with a better gradient (such as rT3 509) is selected to prepare a monoclonal phage supernatant, and the positive clones are identified by Phage-ELISA to obtain positive sequences.

[0114] After library construction and screening, multiple variable region sequences were identified. The diagnostic performance evaluation results of some of the sequences and samples are shown in Table 3:

[0115] Antibody 1: its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1; its light chain variable region has the amino acid sequence shown in SEQ ID NO: 2.

[0116]

[0117] The results show that the effects of Antibody 1, Antibody 2, and Antibody 3 are better than the positive control, and subsequent experiments are carried out based on this.

[0118] Example 4 Expression and purification of the recombinant monoclonal antibody of the present invention

[0119] 1. Transient Expression and Purification

[0120] The gene sequencing results of Antibody 1, Antibody 2, and Antibody 3 were constructed by PCR, constructed into the pCMV3 vector, and transfected into HEK293F cells. 25 ml of the transfection system was expressed during transient transfection, and then a small sample was purified using a gravity SPA column. The purified antibody was detected by SDS-PAGE and the purity was >95%, which was then used for preliminary evaluation of the subsequent antibody.

[0121] (1) Preliminary evaluation of antibody-coated magnetic beads

[0122] Antibodies 1, 2, and 3 were used as test kit components, and 25 clinical samples within the test range were randomly selected for testing. Correlation analysis was performed between the test concentrations and the hospital-assigned clinical samples. Antibody 3 showed poor correlation and some outliers, and was not evaluated further.

[0123] (2) Accelerated thermal stability test of Antibody 1 and Antibody 2:

[0124] Antibody-coated magnetic beads were subjected to accelerated thermal stability testing at 37°C for 7 days. Five clinical samples were tested on the machine. The test results are shown in Tables 4 and 5:

[0125]

[0126]

[0127] Antibody 2 has poorer thermal stability than Antibody 1, so subsequent evaluation will focus on Antibody 1. Stable cell lines will be constructed.

[0128] (3) Construction of stable cell lines:

[0129] The heavy and light chain antibody gene sequences were double-digested with XmaJI / BstZ17I and EcoRV / PacI, respectively, and then ligated into the expression vector pCHO 1.0. The recombinant plasmids were transformed into competent DH5α cells, and positive clones were selected using screening plates for sequencing and plasmid extraction. The extracted plasmids were linearized with RruI and transfected into FD-CHOS cells.

[0130] 48 hours after transfection, ELISA was used to detect antibody expression. Positive cell pools were then screened using a stress culture medium containing 200nM MTX and 20μg / ml Puromycin. After cell viability recovered from the first stress cycle, the screening concentration was increased (1000MTX and 50μg / ml Puromycin) for a second round of stress. Once cell viability recovered after the second round of stress, the stable cell line pool was constructed. The stable cell line pool was then used for monoclonal screening. Using a 96-well plate, 24-well plate, 6-well plate, 6-well plate, and SF125 shake flask, cell lines with stable, high expression of the monoclonal antibody were identified.

[0131] (4) Recombinant antibody expression

[0132] The selected stable high-expressing monoclonal cell lines were revived and cultured at a rate of 5×10 5 Cells were passaged for two generations at a density of 10 cells / ml. Evaluation began when the cell viability recovered to no less than 95%. The feeding strategy was as follows: feed was added on days 1, 3, 5, 7, 9, and 11 (the feeds included feed A and feed B, both of which were finished feeds, with feed A added at a ratio of 5% of the cell supernatant and feed B added at a ratio of 0.5% of the cell supernatant). Sugar was added on days 4, 6, 8, 10, and 12 to maintain a glucose concentration of 5 g / L. Cells were then cultured until the cell viability dropped below 70%, at which point the cell supernatant containing the rT3 recombinant monoclonal antibody was harvested.

[0133] (5) Antibody SPA purification and SDS-PAGE identification

[0134] The chromatography workstation used to purify 200 ml of cell supernatant was loaded into a 10 ml SPA column, with a flow rate of 8 mL / min. The column was rinsed with 0.02 mol / L PBS (PH = 7.2-7.4) equilibration buffer. The sample was loaded at a flow rate of 4 mL / min. When the UV peak rose, flow-through was collected. Reequilibration: the flow rate was set to 8 mL / min. The column was rinsed again with 0.02 mol / L PBS (PH = 7.2-7.4) equilibration buffer until the UV peak dropped to the baseline, and flow-through was stopped. Dissociation: The target protein was dissociated with 0.2 mol / L Gly + 0.15 mol / L NaCl (PH = 2.7) dissociation buffer. When the UV peak began to rise, the target protein was collected. When the UV peak dropped to the baseline, the target protein was stopped and 1 mol / L Tris (PH = 8.5) was added to the collection tube to neutralize it to pH 7. The collected proteins were combined and detected by SDS-PAGE. Figure 6 This indicates that the purified antibody has high purity and concentration, meeting the requirements.

[0135] Example 5 Application of the kit for the rT3 recombinant rabbit monoclonal antibody prepared by the present invention

[0136] The rT3 recombinant rabbit monoclonal antibody prepared in this invention is used as a magnetic bead-coated antibody in a kit, along with other components, to detect antigens in samples. Specific performance evaluations are as follows:

[0137] 1. Accuracy determination: 50 clinical samples with the same value as a mainstream commercial kit on the market were used for testing. The rT3 concentration in the samples was calculated by the system and the correlation analysis was performed with the reference manufacturer. The results are shown in Figure 7 According to the test results, the linear equation is: y = 0.9727x-0.0428, and the correlation coefficient R 2 = 0.9789, indicating that the rT3 recombinant rabbit monoclonal antibody provided by the present invention has a high accuracy in detecting rT3 clinical samples.

[0138] 2. Sensitivity performance evaluation:

[0139] Five clinical samples close to 0 were selected, and each sample was repeated 3 times for a total of 4 days, obtaining 60 data points. Data verification and result analysis were performed according to the CLSI EP17-A classic method. The LoB values of clinical 0 samples in the three batches of test kits were all less than 0.15nmol / L, and all were detected within the blank limit, indicating high sensitivity.

[0140] 3. Recovery performance evaluation:

[0141] Three high-value samples were selected and added to three low-value / matrix samples at a ratio of 1:9 to prepare recovery samples. The volume of the high-value sample added should not exceed 10% of the total volume. Each recovery sample was tested three times, and the average value was calculated to determine the recovery rate. The recovery rates were between 95% and 105%, meeting the required recovery accuracy.

[0142] 4. Linear analysis:

[0143] We selected clinical high-value samples with rT3 concentrations close to 130% of the upper limit of the expected linear range, and 8 clinical low-value samples with concentrations close to 0 or the lowest value that can be obtained clinically, and mixed them in different proportions to obtain 9 linear samples with different concentrations. Finally, we calculated the deviation between the test results and the theoretical concentration ( Figure 8 ). The results show that the linear regression coefficient R 2 >0.99, linear sample deviation is less than 10%.

[0144] 5. Precision Assessment: This study evaluated intra-batch precision. Three concentration levels (high, medium, and low) were tested using the same batch of reagents. Each sample in each batch was replicated 20 times, and the mean, SD, and coefficient of variation (CV) were calculated. The intra-batch coefficient of variation (CV) for all three levels was less than 5%, as shown in Table 6, demonstrating good reproducibility.

[0145]

[0146] 6. Stability assessment:

[0147] The rT3 antibody-coated magnetic microparticle suspension was stored at 37°C for 7 days and then subjected to relevant performance evaluation tests. The specific results are shown in Table 7. The performance indicators of the reagent after heat treatment all met the requirements, and there was no deviation in the test results.

[0148]

[0149] 7. Cross-substance evaluation: The cross-substance was prepared into the test concentration using hormone-free serum, and the cross-substance was tested. The results are shown in Table 8. The cross-substance rates at the test concentration were all below 0.5%, meeting the requirements of the test kit.

[0150]

[0151] 8. Matrix effect assessment:

[0152] At least 20 clinical samples of the day were evenly distributed within the linear range. The matrix preparations, such as calibrator matrix, serum pool, and quality control matrix, were then prepared based on the desired matrix, including commercial quality control matrix. The concentration range of the prepared samples should be evenly distributed within the linear range of the kit. Based on the evaluation results, the matrix preparations to be evaluated were all within their 95% confidence intervals in the calibrator matrix, serum pool, and quality control matrix, indicating that there is no matrix effect in the application of this antibody.

[0153] 9. Evaluation of the difference between new and old samples:

[0154] Twenty clinical samples within the linear range were randomly selected and evaluated on the same instrument using the same kit on days 0, 5, 7, and 10 after sampling. The rT3 concentrations in the samples were back-calculated on different sampling days. The correlation coefficient R^2 between samples on different days was greater than 99%. There was no difference in the application of this antibody when testing new and old samples.

[0155] The above results indicate that the recombinant monoclonal antibody prepared by the present invention has achieved a level highly consistent with the detection results of mainstream manufacturers in the current market, and exhibits advantages such as high sensitivity and excellent stability. When used as a detection antibody in a test kit, the performance of each detection item meets the requirements of the test kit according to the evaluation, which plays an important role in subsequent applications and clinical diagnosis.

[0156] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. rT3 monoclonal antibody, characterized in that The amino acid sequences of the CDR region of its heavy chain are GFSLDDYT, IYDNGRT, and ARKGAFGPDNAINSL; The amino acid sequences of the CDR region of its light chain are QSVYNNNQ, DAS and LGRYSSGDRRA.

2. The rT3 monoclonal antibody according to claim 1, characterized in that The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 1; The amino acid sequence of its light chain variable region is shown in SEQ ID NO:

2.

3. The rT3 monoclonal antibody according to claim 1 or 2, characterized in that The heavy chain constant region is of rabbit IgG1 subtype, and the light chain constant region is of rabbit K1 type.

4. A nucleic acid encoding the rT3 monoclonal antibody according to any one of claims 1 to 3.

5. The nucleic acid according to claim 4, characterized in that A nucleic acid encoding the heavy chain variable region of the rT3 monoclonal antibody, having a nucleotide sequence as shown in SEQ ID NO: 3; The nucleic acid encoding the light chain variable region of the rT3 monoclonal antibody has the nucleotide sequence shown in SEQ ID NO:

4.

6. A recombinant vector, characterized in that It comprises a vector backbone and the nucleic acid according to claim 4 or 5.

7. The recombinant vector according to claim 6, characterized in that The vector backbone is pCHO1.

0.

8. A host cell transformed or transfected with the recombinant vector according to claim 6 or 7.

9. A labeled antibody, characterized in that The method comprises a marker and the rT3 monoclonal antibody according to any one of claims 1 to 3; the marker is selected from a chemical marker or a biological marker.

10. The labeled antibody according to claim 9, characterized in that The biological markers include biotin, avidin or enzymes; the chemical labels include isotopes.

11. A conjugate, characterized in that The method comprises a coupling medium and the rT3 monoclonal antibody according to any one of claims 1 to 3; the coupling medium is a solid medium or a semi-solid medium.

12. A method for preparing rT3 monoclonal antibody, characterized in that: The method comprises culturing the host cell according to claim 8 to obtain the rT3 monoclonal antibody.

13. Use of any one of the following I) to VII) in the preparation of a product for detecting rT3 levels in serum: 1), the rT3 monoclonal antibody according to any one of claims 1 to 3; II), the nucleic acid according to claim 4 or 5; III), the recombinant vector according to claim 6 or 7; IV), the host cell according to claim 8; V), the labeled antibody according to claim 9 or 10; VI), the conjugate according to claim 11; VII), a culture containing rT3 monoclonal antibody obtained by the preparation method according to claim 12.

14. A product for detecting rT3 in serum, characterized in that: It includes any one or more of the following i) to iv): i), the rT3 monoclonal antibody according to any one of claims 1 to 3; ii), the labeled antibody according to claim 9 or 10; iii) the conjugate according to claim 11; iv) A culture containing rT3 monoclonal antibody obtained by the preparation method according to claim 12.

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