Anti-testosterone antibody or functional fragment thereof and application thereof

Abstract

The invention belongs to the technical field of biology, particularly relates to an anti-testosterone antibody or a functional fragment thereof and application of the anti-testosterone antibody or the functional fragment thereof, and particularly discloses an anti-human T antibody or an antigen binding part antibody thereof, which has high affinity and stable performance on T and has great significance on obtaining a correct diagnosis result in the early stage of prostatic cancer.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to anti-testosterone antibodies or their functional fragments and their applications. Background Technology

[0002] Testosterone (T), also known as testosterone, is a steroid hormone secreted by the testes in men and the ovaries in women, with the adrenal glands also secreting small amounts. It is the primary male sex hormone and anabolic steroid. In both men and women, it has significant effects on health, including enhancing libido, strength, immune function, and combating osteoporosis. Statistics show that adult men secrete 20 times more testosterone than adult women.

[0003] Testosterone's effects can be divided into anabolism and masculinizing effects. Anabolism includes increased muscle mass and strength, increased bone density and strength, stimulation of linear growth and skeletal maturation, etc. Masculinizing effects refer to male secondary sexual characteristics, such as the maturation of sex organs, voice deepening, and the growth of beard and armpit hair. The effects of testosterone differ between men and women, and can be distinguished by the timing of natural testosterone production, primarily influenced by the amount and duration of free testosterone transport. Testosterone is a hormone produced by the body itself, and unless there is a pathological reason or special circumstances, no additional supplementation is necessary. Excessive testosterone levels can have adverse effects; in men, it can lead to precocious puberty and testicular diseases, while in women, it can cause infertility.

[0004] In addition, testosterone is one of the important sex hormones in men, and the prostate is androgen-dependent. Androgen levels are closely related to prostate lesions. Therefore, the diagnosis of testosterone levels plays an important role in the early diagnosis of prostate cancer.

[0005] Currently, most T-cell antibody diagnoses are based on mouse monoclonal antibodies. Sheep, being larger than mice and rabbits, are suitable for producing large quantities of antiserum, and therefore, in the field of biotechnology, sheep are widely used to produce polyclonal antibodies. Existing research shows that sheep have higher immunosensitivity and can recognize a wider range of epitopes than mice and rabbits. Furthermore, comparative studies have shown that sheep antibodies generally have higher affinity and specificity than standard mouse, rat, and rabbit antibodies (cited in Hybridoma. 1999 Apr; 18(2):183-91). The ultra-high affinity and broad epitope recognition characteristics of sheep monoclonal antibodies give them a significant advantage in detecting very low concentrations of small molecules, such as hormones and drug residues. Phage display technology provides a powerful tool for screening novel proteins (such as antibodies) that bind to specific ligands. In phage display technology, by constructing a large library containing a large number of different antibody sequences, it becomes possible to rapidly screen and isolate antibody sequences that can bind to the target antigen with high affinity. In the developed monovalent phage display system, nucleic acids encoding different antibodies are fused with nucleic acid sequences encoding viral capsid proteins (such as PIII protein). In such a display system, the antibody gene fusion is expressed at low levels in bacterial cells, and wild-type PIII protein is also expressed in the same bacterial cell, thus ensuring the infectivity of newly generated phage particles. Many documents have disclosed methods for constructing such libraries and screening for target antibodies, such as (Biochemistry. 1991 Nov 12; 30(45):10832-8.; J BiolChem. 1997 Apr 18; 272(16):10678-84.; Methods Enzymol. 2016; 580:45-87.; US5723286A; US5427908A, etc.). This method of expressing polypeptides or antibody fragments on the surface of filamentous phages is the most important feature of phage display technology. A library is formed by a large number of phage particles displaying diverse sequences of polypeptides or antibody fragments. Library construction can be achieved by cloning relevant antibody gene families, such as the BCR gene extracted after immunizing animals with specific antigens, or by isolating all BCR components from B cells of immunized or unimmunized individuals. After successful library construction, antibodies with desired characteristics are obtained through various screening methods. Using phage display technology to prepare antibodies has unique advantages. This technology allows for the development of target antibody sequences in a short time without the use of animals, for example, using commercially available natural libraries. Furthermore, while hybridoma technology, commonly used in antibody development, requires the development of appropriate myeloma cells for different species, phage display technology is not species-restricted.In addition, for some antigens that have high sequence homology with endogenous proteins in animals, it may be impossible to elicit an immune response in the animal, making it difficult to generate the target B cells. In this case, hybridoma technology will not be able to develop and obtain the target antibody; however, phage display technology may be able to bypass the problem of immune tolerance and screen for suitable antibody sequences. Summary of the Invention

[0006] This invention involves preparing a T immunogen, immunizing 6-12 month old female sheep, and after 6-9 immunizations, collecting fresh peripheral blood with sufficient titers for PBMC isolation and gene library construction. Through phage display screening, this invention provides a sheep monoclonal antibody with a high recognition reactivity to the T protein.

[0007] Specifically, anti-human T antibodies or their antigen-binding portions include:

[0008] A sheep monoclonal antibody against human T cells or its functional fragments, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein HCDR1 to HCDR3 are amino acid sequences consistent with HCDR1 to HCDR3 in the heavy chain variable region shown in SEQ ID NO:1, and LCDR1 to LCDR3 are amino acid sequences consistent with LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO:2.

[0009] Preferably, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a Kabat, Chothia, IMGT, AbM, or Contact system.

[0010] Preferably, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0011] The HCDR1 is shown in SEQ ID NO:3;

[0012] The HCDR2 is shown in SEQ ID NO:4;

[0013] The HCDR3 is shown in SEQ ID NO:5;

[0014] The LCDR1 is shown in SEQ ID NO:6;

[0015] The LCDR2 is shown in SEQ ID NO:7;

[0016] And the LCDR3 is as shown in SEQ ID NO:8.

[0017] In this paper, the terms "variable region" or "CDR" refer to the highly variable region of the heavy and light chains of an immunoglobulin, which contains one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes.

[0018] In this document, heavy chain variable regions (heavy chain complementarity-determining regions) are denoted by "HCDR," which includes HCDR1, HCDR2, and HCDR3; light chain complementarity-determining regions are denoted by "LCDR," which includes LCDR1, LCDR2, and LCDR3. Commonly used CDR labeling methods in the art include the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the Kabat database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. This disclosure uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods are also within the scope of this invention.

[0019] In this article, the "backbone region" or "FR" region includes the heavy chain backbone region and the light chain backbone region, which refers to the region in the antibody heavy chain variable region (which can be represented as VH) and light chain variable region (which can be represented as VL) excluding the CDR. The heavy chain backbone region is represented by "HFR" and can be further subdivided into adjacent regions separated by CDR, including the HFR1, HFR2, HFR3 and HFR4 backbone regions; the light chain backbone region is represented by "LFR" and can be further subdivided into adjacent regions separated by CDR, including the LFR1, LFR2, LFR3 and LFR4 backbone regions.

[0020] Preferably, the antibody or its functional fragment further comprises the heavy chain variable region backbone regions HFR1, HFR2, HFR3, and HFR4, and the light chain variable region backbone regions LFR1, LFR2, LFR3, and LFR4.

[0021] Wherein, HFR1 is an amino acid sequence as shown in EQ ID NO:9 or an amino acid sequence having at least 80% homology with it;

[0022] The HFR2 is an amino acid sequence as shown in EQ ID NO:10 or an amino acid sequence having at least 80% homology with it;

[0023] The HFR3 is an amino acid sequence as shown in EQ ID NO:11 or an amino acid sequence having at least 80% homology with it;

[0024] The HFR4 is an amino acid sequence as shown in EQ ID NO:12 or an amino acid sequence having at least 80% homology with it;

[0025] The LFR1 is an amino acid sequence as shown in EQ ID NO:13 or an amino acid sequence having at least 80% homology with it;

[0026] The LFR2 is an amino acid sequence as shown in EQ ID NO:14 or an amino acid sequence that is at least 80% homologous to it;

[0027] The LFR3 is an amino acid sequence as shown in EQ ID NO:15 or an amino acid sequence having at least 80% homology with it;

[0028] The LFR4 is an amino acid sequence as shown in EQ ID NO:16 or an amino acid sequence that has at least 80% homology with it.

[0029] In this paper, the variable region of the heavy chain is obtained by connecting the following CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the variable region of the light chain is obtained by connecting the following CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0030] It should be noted that, in other embodiments, the amino acid sequences of each backbone region of the antibody or its functional fragment provided in this disclosure may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the corresponding backbone regions (SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, or 16). In optional embodiments, the antibody or its functional fragment has a KD ≤ 10. -7 M, KD≤10 -8 M, KD≤10 -9 M, KD≤10 -10 M or KD≤10 -11 The affinity of T is combined with human T.

[0031] Preferably, it also includes a constant region;

[0032] Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region;

[0033] Optionally, the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, T, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0034] Optionally, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans;

[0035] Optionally, the heavy chain constant region is an amino acid sequence as shown in EQ ID NO:17 or an amino acid sequence having at least 80% homology therewith, and the light chain constant region is an amino acid sequence as shown in EQ ID NO:18 or an amino acid sequence having at least 80% homology therewith.

[0036] Preferably, the functional fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

[0037] The functional fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand from the content of this disclosure that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this disclosure, those skilled in the art can readily obtain the aforementioned functional fragments.

[0038] The functional fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by automated peptide synthesizers, such as those sold by AppliedBioSystems.

[0039] On the other hand, the present invention also discloses an antibody conjugate comprising the antibody or a functional fragment thereof and a conjugated portion thereof;

[0040] Optionally, the coupling portion is selected from purified tags or detectable labels, such as colloidal gold, radioactive labels, luminescent substances, colored substances, enzymes such as fluorescent labels, chromophore labels, electron-dense labels such as radioactive isotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin / antibiotin protein, and one or more of spin labels;

[0041] Optionally, the coupling portion is selected from solid-phase carriers, such as magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0042] On the other hand, the present invention discloses a nucleic acid, characterized in that the nucleic acid encodes the antibody or a functional fragment thereof.

[0043] Nucleic acids are typically RNA or DNA, and nucleic acid molecules can be single-stranded or double-stranded. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence. DNA nucleic acids are used when they are ligated into a vector.

[0044] Some embodiments of this disclosure also provide a carrier containing the aforementioned nucleic acid molecules.

[0045] Some embodiments of this disclosure also provide cells containing the above-described carrier.

[0046] Some embodiments of this disclosure also provide a method for preparing an antibody or a functional fragment thereof, comprising: culturing cells as described above, and separating and purifying the antibody or a functional fragment thereof from the culture product.

[0047] Based on the amino acid sequence of the antibody or its functional fragment in this disclosure, those skilled in the art will readily conceive of using genetic engineering or other techniques (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment, for example, by isolating and purifying the antibody or its functional fragment from the culture product of a recombinant cell capable of recombinantly expressing the antibody or its functional fragment as described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antibody or its functional fragment of this disclosure, it falls within the protection scope of this disclosure.

[0048] On the other hand, the present invention discloses a detection reagent or kit, the reagent or kit comprising the antibody or its functional fragment, and the antibody-drug conjugate.

[0049] In optional embodiments, the blocking agent included in the kit may be in the form of a liquid solution, attached to a solid support, or a dry powder. When the blocking agent is a liquid solution, the liquid solution may be an aqueous solution. When the immunoblocking agent is attached to a solid support, the preferred solid support may be a chromatography medium such as a membrane, test strip, plastic beads or plates, or a microscope slide. When the blocking agent is a dry powder, the powder can be reconstituted by adding a suitable solvent.

[0050] Preferably, the kit is a chemiluminescence detection kit.

[0051] On the other hand, the present invention discloses the use of the antibody or its functional fragment, the antibody conjugate, or the reagent or kit in the preparation of testosterone protein detection products.

[0052] On the other hand, the present invention discloses a method for detecting testosterone protein in a sample, the method comprising:

[0053] a) Under conditions sufficient to induce an antibody / antigen binding reaction, the testosterone protein antigen in the test sample is contacted with the antibody or its functional fragment, the antibody-conjugate, or the reagent or kit to form an immune complex; and

[0054] b) Detect the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample;

[0055] Optionally, in step a), the immune complex further includes a second antibody, which binds to the antibody or antigen-binding fragment;

[0056] Optionally, in step a), the immune complex further includes a second antibody that binds to the testosterone protein antigen.

[0057] Beneficial effects

[0058] The anti-T goat monoclonal antibody disclosed herein exhibits high affinity, high reactivity, and high sensitivity or specificity for human T cells. It demonstrates good specificity and high affinity in immunoassay, providing a detection method for the immunodiagnosis of T cells. Therefore, the anti-T antibody, immunoassay reagents, and kits disclosed herein all possess excellent practical performance and broad market application prospects. Attached Figure Description

[0059] Figure 1 The antibody gel image disclosed in this invention shows that the left side is the marker and the right side is the purified antibody reduction band.

[0060] Figure 2 Results of experiments using enzyme-linked immunosorbent assay (ELISA) to detect the interaction between antibodies and human T antigen;

[0061] Figure 3 Clinical sample test results of the antibody described in this invention were identified using a chemiluminescence method. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions in the embodiments of this disclosure will be clearly and completely described below. Where specific conditions are not specified in the examples or embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0064] Unless otherwise specified, the practice of this disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994) and "Current Protocols in Immunology" (JEColigan et al., ed., 2011), each of which is explicitly incorporated herein by reference.

[0065] Example 1: Preparation of T antibody

[0066] 1. Preparation of human T immunogen

[0067] Human T-cell immunogen was obtained by digesting human T-cells with pepsin in sodium citrate buffer solution at 25-37℃ and pH 3.5 for 2 hours. The Fc terminus was then purified using a His tag to obtain T-Fc terminus with a purity greater than 75%. This T-Fc terminus was constructed into the pcDNA3.1(+) vector and transfected into HEK293 cells (plasmid concentration 1 μg / mL). After culturing for 5 days, the cells were purified using a Ni Excel column to obtain the human T-Fc immunogen.

[0068] 2. Animal immunization

[0069] Three healthy female sheep aged 6-12 months were selected for immunization. For the initial immunization, 250 μg of human T-Fc immunogen was mixed with Freund's complete adjuvant at a 1:1 ratio and emulsified, and administered at multiple sites including the left / right axilla, around the cervical lymph nodes, and the shoulder and back. Subsequent booster immunizations were given at 3-week intervals using Freund's incomplete adjuvant. Serum titers were measured after each immunization, and a titer >1 million was considered acceptable (6-9 immunizations). One week after the final immunization, peripheral blood was collected from the sheep, and PBMCs were isolated.

[0070] Three healthy female sheep aged 6-12 months were selected for immunization. For the initial immunization, 250 μg of human T-Fc immunogen was mixed with Freund's complete adjuvant at a 1:1 ratio and emulsified, and administered at multiple sites including the left / right axilla, around the cervical lymph nodes, and the shoulder and back. Subsequent booster immunizations were given at 3-week intervals using Freund's incomplete adjuvant. Serum titers were measured after each immunization, and a titer >1 million was considered acceptable (6-9 immunizations). One week after the final immunization, peripheral blood was collected from the sheep, and PBMCs were isolated.

[0071] 3. Preparation of peripheral blood PBMCs

[0072] Select immunized sheep with high titers and collect 50 mL of fresh blood. Dilute the blood sample 1:1 with PBS. Take five 50 mL centrifuge tubes and add 20 mL of Histopaque (Sigma) separation medium to each. Carefully add 20 mL of diluted blood sample to each tube above the surface of the Histopaque separation medium. Centrifuge at 400 g for 35 minutes at room temperature to separate PBMCs. After centrifugation, granulocytes, platelets, and erythrocytes will pass through the separation medium and remain at the bottom of the centrifuge tube, while PBMCs will remain on top of the Histopaque separation medium. Wash the collected PBMCs twice with PBS and collect the cells by centrifugation at 300 g.

[0073] 4. Construction of phage display library

[0074] Using freshly collected sheep PBMC cells, add 1 mL of TRIzol per 1E7 cells. TMReagent reagent. Invert and mix repeatedly until all cells are lysed and no obvious precipitate appears. Incubate at room temperature for 10 minutes. Add 1 / 5 volume of chloroform and shake vigorously for 1 minute to mix thoroughly. Centrifuge the mixture at 12000g for 10 minutes at 4°C and collect the supernatant. Carefully add an equal volume of isopropanol to the supernatant, invert and mix thoroughly, and incubate at room temperature for 30 minutes to precipitate RNA. Centrifuge at 12000g for 10 minutes at 4°C to collect the RNA precipitate. Wash the RNA precipitate with 800µl of 75% ethanol, evaporate the ethanol, dissolve the RNA in 50µl of RNase-free water, and quantify. PrimeScript was used. TM II. Reverse transcription using Reverse Transcriptase (2690A) was performed to prepare cDNA according to the manufacturer's instructions. A sheep scFv immunogen library targeting the human T-Fc immunogen was constructed using publicly available experimental methods. (Reference: J Immunol Methods. 2000 Mar 6; 236(1-2):133-46; Phage Display—A Practical Approach. Edited by T. Clackson and H. B. Lowman, Oxford University Press, Oxford, UK; 2004;)

[0075] The PCR primers for the cloned sheep V region gene used in this invention are shown in SEQ ID NO:19-39, wherein VHF1, VHF2, VHF3, VHF4, VHR1, VHR2, VHR3, VLF1, VLF2, VLR1, VLR2, VLR3, VLR4, VLR5, VLR6, VLR7, VKF1, VKF2, VKR1, VKR2, and VKR3 correspond to SEQ ID NO:19-39 in sequence.

[0076] Phanta Max Super-Fidelity DNA Polymerase (P505-d3) produced by Nanjing Novizan Biotechnology Co., Ltd. was used for the amplification of various gene fragments and the splicing and assembly of scFv.

[0077] PCR amplification conditions were as follows: 95℃ pre-denaturation for 3 min (1 cycle); 95℃ denaturation for 13 s, 56℃ annealing for 15 s, 72℃ extension for 30 s (30 cycles); 72℃ final extension for 5 min (1 cycle). All amplified fragments were purified and recovered using a DNA agarose gel purification kit (OMEGA, D2500-02). The spliced ​​scFv fragments were digested with SfiI restriction endonuclease (NEB, R0123L) and then ligated into the pComb3X phage vector (addgene, #63890). The ligation product was purified using a DNA purification kit (OMEGA, D6492-02) and then used for electroporation transformation of TG1 competent cells (Agilent, 200123). After recovery culture, all transformed TG1 cells were plated on plates containing 100 μg / mL Amp antibiotic and cultured overnight. The resulting culture was used as a gene library. The TG1 gene library was infected with M13K07 helper phage (NEB, N0315S) and cultured overnight. The culture supernatant was then precipitated with polyethylene glycol to prepare the phage display library.

[0078] 5. Phage-antibody washing and clone identification

[0079] To obtain the suitable antibody described in this invention, the antigen human T-Fc immunogen was diluted with PBS to 100 ug / ml, 10 ug / ml, 1 ug / ml and 0.1 ug / ml, respectively, and coated onto immunotubes (NUNC MaxiSorp). TM 5E12 phage-scFv particles were incubated with the coated antigen and washed with PBS containing 0.1% Tween 20. The concentration of the coated antigen was gradually reduced and the number of washes increased with each wash. The bound target phages were eluted with 10 mM HCl and neutralized with 1 M Tris-HCl, pH 8.3. The eluted target phages were then used to infect fresh exponentially proliferating TG1 cells and plated on YTAG plates (1.6% tryptone, 1% yeast extract, 0.5% sodium chloride, 100 μg / mL ampicillin, 2% glucose) to obtain single clones. Single clones were selected and cultured to an OD600 of 0.5, then infected with M13K07 helper phages (NEB, N0315S) and cultured overnight in YTAK medium (1.6% tryptone, 1% yeast extract, 0.5% sodium chloride, 100 μg / mL ampicillin, 50 μg / mL kanamycin). The culture supernatant contained a large number of phage-scFv single-clone particles. This culture supernatant was used for ELISA to identify clones that specifically bind to human T-Fc immunogen. The gene coding sequence of this phage-scFv was obtained by gene sequencing.

[0080] 6. Antibody expression and purification

[0081] 1. The gene sequence was synthesized by Shanghai Sangon Biotech and constructed into the pCDNA3.4 vector;

[0082] 2. Resuscitate and passage Expi293 cells, seeding them at a density of 1E6 24 hours before transfection;

[0083] 3. On the day of transfection, when the cell density reaches 2-2.5E6 and the cell viability is greater than 98%, transfection can begin.

[0084] 4. The plasmid addition amount is 1 μg / ml, the heavy to light chain ratio is 1:1, and the plasmid to PEI ratio is 1:3. After mixing, allow to stand at room temperature.

[0085] 5. Let stand for 15 minutes, then add to the prepared cell culture medium. Incubate at 37°C and 120 rpm for suspension culture.

[0086] 6. Add supplementary material 1 after 24 hours, at a rate of 1%;

[0087] 7. Add supplementary material 2 after 48 hours, at a rate of 2%;

[0088] 8. After 5 days of fermentation, when the cell viability is greater than 70%, centrifuge at 5000 rpm and 4℃ to collect the sample;

[0089] 9. Prepare column G and equilibrate 5 column volumes with PBS;

[0090] 10. Flow through the column at a flow rate of 2 ml / min. After the sample has flowed through the column, equilibrate with 5 column volumes using PBS.

[0091] 11. Elute with 0.5M glycine at pH 2.7 and dialyze into PBS buffer;

[0092] 12. After concentration using ultrafiltration, quantification was performed using the BCA method, yielding a final T-mAb antibody concentration of 5 mg / mL. The results of the reduced SDS-PAGE are shown below. Figure 1 As shown.

[0093] Example 2 Antibody Performance Verification

[0094] This embodiment verifies the binding performance of the antibody of the present invention through the interaction between the antibody and human T-Fc.

[0095] The specific operating steps are as follows: Dilute the antibody to 1 μg / mL using coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer) and coat it onto a standard 96-well ELISA plate. Modify the purified human T-Fc obtained after recombinant expression with biotinylation reagent (commercially available reagent). Dilute the biotinylated human T-Fc to the target concentrations (0.3 ng / ml, 0.8 ng / ml, 2.5 ng / ml, ..., 200 ng / ml). Add the specific concentration of human T-Fc (100 μl per well) to the ELISA wells coated with the target antibody and incubate at 37°C for 30 min. After washing the plate with washing buffer (150 mM NaCl solution containing 0.05% Tween-20), add 100 μl of streptavidin-horseradish peroxidase conjugate (1:5000 dilution, commercially available reagent) to each well and incubate at 37°C for 15 min. Finally, the sample was incubated with ELISA colorimetric solution and the OD450 absorbance was detected using a microplate reader.

[0096] The results are shown in Table 1 and Figure 2 As shown, the results of the enzyme-linked immunosorbent assay (ELISA) were used to detect the interaction between the antibody and recombinant expressed and purified human T-Fc. The horizontal axis indicates the concentration of human T-Fc used, and the vertical axis indicates the detection result of the ELISA experiment, i.e., the absorbance value (OD450) detected by the spectrophotometer at 450 nm. A higher OD450 value indicates a stronger detected interaction. The results show that the antibody interacts strongly with human T-Fc, and the antibody of this invention is suitable for detecting human T-Fc.

[0097] Table 1. Results of linear experiments

[0098] Human T-Fc (ng / mL) T-mab 200 2.297 66.7 2.111 22.2 1.706 7.4 0.98 2.5 0.446 0.8 0.185 0.3 0.109 0 0.042

[0099] Example 3: T-mAb antibody chemiluminescence functional detection

[0100] To verify the effectiveness of the purified T antibody in immunoassays, current antibody detection methods include immunoturbidimetry, immunochromatography, and chemiluminescence. Chemiluminescence has the most stringent requirements on antibody performance; therefore, this embodiment selects a competitive chemiluminescence assay. Chemiluminescence kits can be purchased commercially.

[0101] In this embodiment, the antibody purified in step 1 was selected for the experiment.

[0102] The assay reagents include R1, R2, and a magnetic bead solution.

[0103] R1 includes: biotin-labeled T-mAb, concentration: 1ug / ml;

[0104] R2 comprises: avidin-labeled magnetic beads at a concentration of 0.1 ug / ml.

[0105] The specific operating steps are as follows:

[0106] 1. Experimental preparation: 5 ml each of R1 and R2 working solutions, 4 ml of magnetic bead solution, calibrator, and T-mAb.

[0107] 2. Place each component into the kit separately, and then place the kit into the EXI1800 fully automated chemiluminescence immunoassay analyzer (from Zhongyuan Huiji Biotechnology Co., Ltd.). Select item T (sample 10ul, R160ul, R2125ul, magnetic bead solution 30ul) for the experiment.

[0108] Clinical sample: 40 randomized clinical samples, which can be collected from various hospitals.

[0109] Table 2 Clinical Sample Test Results

[0110]

[0111]

[0112] The results are shown in Table 2 and Figure 3 As shown, the detection results of the reagent of the present invention are highly consistent with the clinical results, indicating that the antibody of the present invention has excellent clinical detection performance.

[0113] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An anti-testosterone antibody or a functional fragment thereof, comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1-HCDR3 are amino acid sequences consistent with HCDR1-HCDR3 of the heavy chain variable region shown in SEQ ID NO:1, and the LCDR1-LCDR3 are amino acid sequences consistent with LCDR1-LCDR3 of the light chain variable region shown in SEQ ID NO:

2.

2. The antibody or functional fragment thereof according to claim 1, characterized in that: The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat, Chothia, IMGT, AbM or Contact systems.

3. The antibody or functional fragment thereof according to claim 2, characterized in that: The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system, Wherein, the HCDR1 is shown in SEQ ID NO:3; The HCDR2 is shown in SEQ ID NO:4; The HCDR3 is shown in SEQ ID NO:5; The LCDR1 is shown in SEQ ID NO:6; The LCDR2 is shown in SEQ ID NO:7; And the LCDR3 is shown in SEQ ID NO:

8.

4. The antibody or functional fragment thereof according to any one of claims 1 to 3, characterized in that: The antibody or its functional fragment further comprises the framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region, and the framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region, Wherein, the HFR1 is an amino acid sequence as shown in EQ ID NO:9 or an amino acid sequence having at least 80% homology thereto; The HFR2 is an amino acid sequence as shown in EQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto; The HFR3 is an amino acid sequence as shown in EQ ID NO: 11 or an amino acid sequence having at least 80% homology thereto; The HFR4 is an amino acid sequence as shown in EQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto; The LFR1 is an amino acid sequence as shown in EQ ID NO: 13 or an amino acid sequence having at least 80% homology thereto; The LFR2 is an amino acid sequence as shown in EQ ID NO: 14 or an amino acid sequence having at least 80% homology thereto; The LFR3 is an amino acid sequence as shown in EQ ID NO: 15 or an amino acid sequence having at least 80% homology thereto; The LFR4 is an amino acid sequence as shown in EQ ID NO: 16 or an amino acid sequence having at least 80% homology thereto; Preferably, the antibody or its functional fragment further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, T, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region; Optionally, the species of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans; Preferably, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

5. The antibody or functional fragment thereof according to any one of claims 1 to 4, characterized in that: The heavy chain constant region is an amino acid sequence as shown in EQ ID NO: 17 or an amino acid sequence having at least 80% homology thereto, and the light chain constant region is an amino acid sequence as shown in EQ ID NO: 18 or an amino acid sequence having at least 80% homology thereto.

6. A nucleic acid, characterized in that The nucleic acid encodes the antibody or antigen-binding portion thereof according to any one of claims 1 to 5.

7. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody or its functional fragment according to any one of claims 1 to 5 and a conjugated part conjugated thereto; Optionally, the antibody or functional fragment thereof is labeled with a marker; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the fluorescent dye is selected from fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives; Optionally, the enzyme is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and 6-phosphoglucose deoxygenase; Optionally, the radioisotope is selected from 212 Bi, 13 1I. 111 In, 90 Y. 186 Re, 211 At 125 I. 188 Re, 153 Sm, 213 Bi, 32 P. 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn、 97 Such as 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F; Optionally, the chemiluminescent agent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives; Optionally, the nanoparticle-based marker is selected from nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles; Optionally, the colloid is selected from colloidal metals, disperse dyes, dye-labeled microspheres and latex; Optionally, the colloidal metal is selected from colloidal gold, colloidal silver and colloidal selenium; Optionally, the antibody or functional fragment thereof is coated onto a solid phase; Optionally, the solid phase is selected from microspheres, plates and membranes; Optionally, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

8. A detection reagent or kit, characterized in that: The reagent or kit comprises the antibody or functional fragment thereof according to any one of claims 1 to 5 or the antibody conjugate according to claim 7.

9. Use of the antibody or functional fragment thereof according to any one of claims 1 to 5, the antibody conjugate according to claim 7, or the reagent or kit according to claim 8 in the preparation of a testosterone protein detection product.

10. A method for detecting testosterone protein in a sample, characterized in that: The method comprises: a) contacting the testosterone protein antigen in the test sample with the antibody or functional fragment thereof according to any one of claims 1 to 5, the antibody conjugate according to claim 7, or the reagent or kit according to claim 8 under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample; Optionally, in step a), the immune complex further comprises a second antibody, which binds to the antibody or antigen-binding fragment; Optionally, in step a), the immune complex further comprises a second antibody, and the second antibody binds to the testosterone protein antigen.

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