Anti-human progesterone antibody or functional fragment thereof and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-human progesterone antibody or a functional fragment thereof and application thereof. The antibody sequence disclosed by the invention is stable in antibody performance and high in affinity to progesterone small molecules, and a human progesterone detection chemiluminescence diagnostic reagent developed on the basis of the antibody shows excellent performance for progesterone detection in clinical tests and has important clinical practical value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an anti-human progesterone antibody or a functional fragment thereof and an application thereof.

[0002] The present invention claims priority to application number 202311053318.1 filed on August 21, 2023, the entire contents of which are incorporated herein by reference. Background Art

[0003] The six-item sex hormone test is a routine reproductive system examination item, and many women will use this test when preparing for pregnancy and diagnosing diseases. Among them, progesterone (progesterone, P) is an important steroidal progestogen, which is mainly synthesized by corpus luteum cells and placenta during pregnancy, and is a precursor of testosterone, estrogen and adrenal cortex hormone. Progesterone (P), also known as progesterone, progesterone, or progesterone, is produced at very low levels in both men and women under normal circumstances, while the progesterone level in women during pregnancy is maintained at a higher level. After it is secreted into the blood, it is mainly bound to albumin and sex hormone binding protein to circulate in the body. Progesterone levels are closely related to the development and atrophy of the corpus luteum. Increased progesterone levels can be observed one day before ovulation, and progesterone synthesis increases significantly during the luteal phase. (Quote: Su Guiping. On the application value of serum progesterone determination in predicting the prognosis of early threatened abortion [J]. Contemporary Medical Forum, 2014, 12(2): 178-179.) During the menstrual cycle, the main function of progesterone is to promote the thickening of the endometrium, so that the blood vessels and glands therein proliferate, causing secretion to facilitate the implantation of the fertilized egg (embryo). During pregnancy, progesterone can maintain pregnancy and inhibit the contraction of the uterine muscle layer. Progesterone can also act on the mammary glands, promoting the development of mammary gland alveoli and ducts to prepare for lactation (Quote: Analysis of the value of estradiol, progesterone and β-human chorionic gonadotropin levels in predicting early threatened abortion, Clinical Medical Engineering. 2020, 27(06)). Therefore, the detection of progesterone hormone is of great value for disease diagnosis, fertility diagnosis and prognosis judgment of in vitro fertilization-embryo transplantation.

[0004] In addition, since progesterone can promote the growth and development of animals, regulatory authorities often monitor farmers adding progesterone to animal feed to improve the efficiency of animal farming. The abuse of progesterone will lead to progesterone residues in meat, which will cause a series of food safety problems. Therefore, video detection of progesterone also has important application value.

[0005] Monoclonal antibodies, due to their extremely high spatial structural resolution, make the progesterone detection method based on antigen-antibody immune reaction have many advantages such as strong specificity, high sensitivity, and convenience for large-scale detection on site. Antibodies of different species each have their own unique advantages. Sheep are larger in size than mice and rabbits and are suitable for preparing a large number of antisera. Therefore, in the field of biotechnology, sheep are currently widely used to produce polyclonal antibodies. Existing studies have shown that sheep have higher immune sensitivity and can recognize a wider range of epitopes than mice and rabbits. In addition, comparisons have shown that sheep antibodies usually have higher affinity and specificity than standard mouse, rat, and rabbit antibodies (Hybridoma. 1999Apr; 18 (2): 183-91.). The ultra-high affinity and wide epitope recognition characteristics of sheep monoclonal antibodies make sheep monoclonal antibodies have great advantages in the detection of small molecules at very low concentrations, such as the detection of hormones and drug residues.

[0006] Phage display technology provides a powerful tool for screening new proteins (such as antibodies) that bind to specific ligands. In phage display technology, by constructing a huge library containing a large number of different antibody sequences, it is possible to quickly screen and isolate antibody sequences that can bind to the target antigen with high affinity. In the monovalent phage display system that has been developed, nucleic acids encoding different antibodies are fused with nucleic acid sequences encoding viral coat proteins (such as PIII proteins). In such a display system, the antibody gene fusion will be expressed at low levels in bacterial cells, and the wild-type PIII protein will also be expressed in the same bacterial cells, thereby ensuring the infectivity of the newly generated phage particles. Many materials have disclosed methods for constructing such libraries and screening target antibodies, for example (Biochemistry. 1991 Nov 12; 30(45): 10832-8.; J Biol Chem. 1997 Apr 18; 272(16): 10678-84.; Methods Enzymol. 2016; 580: 45-87.; US5723286A; US5427908A, etc.).

[0007] This way of expressing polypeptides or antibody fragments on the surface of filamentous phage is the most important feature of phage display technology. A large number of phage particles display a large number of polypeptides or antibody fragments of different sequences, which constitute a library. The construction of the library can be achieved by cloning the relevant antibody gene family, such as the BCR gene extracted after immunizing an animal with a specific antigen, or by separating all BCR components from the B cells of immunized or non-immunized people. After the display library is successfully constructed, antibodies with ideal traits are obtained through different screening methods. The use of phage display technology to prepare antibodies has its unique advantages. Using this technology, it is possible to develop and obtain the target antibody sequence in a short time without using animals, such as using natural libraries that have been commercially provided. In addition, the hybridoma technology commonly used in antibody development requires the development of appropriate myeloma cells when developing antibodies of different species, while phage display technology is not restricted by species. In addition, some antigens that have a high degree of sequence homology with endogenous proteins in animals may not be able to stimulate an immune response in the animal, making it difficult to produce the target B cells. In this case, the use of hybridoma technology will not be able to develop the target antibodies; while the use of phage display technology may bypass the problem of immune tolerance and screen for suitable antibody sequences.

[0008] Due to the small molecular weight of progesterone hormone and the difficulty in having a TCR to recognize it, it is still a great challenge to develop high-affinity antibodies specific for progesterone. Therefore, there are few anti-human progesterone antibodies available on the market, and their effectiveness in clinical applications is unknown. Summary of the invention

[0009] The present invention prepares a specific progesterone immunogen, immunizes female sheep, collects fresh peripheral blood of sheep with sufficient titer, separates PBMC, and then constructs a gene library. Through panning and screening with phage display technology, high-quality anti-progesterone sheep monoclonal antibodies are obtained. The immunoassay kit prepared based on the sheep monoclonal antibodies of the present invention shows excellent performance in progesterone detection in clinical tests.

[0010] The present invention provides an antibody having high recognition reactivity to progesterone (P).

[0011] The details are as follows:

[0012] An anti-human progesterone antibody or a functional fragment thereof, comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that the HCDR1 to HCDR3 are amino acid sequences consistent with HCDR1 to HCDR3 of the heavy chain variable region shown in SEQ ID NO:1, and the LCDR1 to LCDR3 are amino acid sequences consistent with LCDR1 to LCDR3 of the light chain variable region shown in SEQ ID NO:2.

[0013] Preferably, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat, Chothia, IMGT, AbM or Contact systems.

[0014] Preferably, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system,

[0015] Wherein, the HCDR1 is shown in SEQ ID NO:3;

[0016] The HCDR2 is shown in SEQ ID NO:4;

[0017] The HCDR3 is shown in SEQ ID NO:5;

[0018] The LCDR1 is shown in SEQ ID NO:6;

[0019] The LCDR2 is shown in SEQ ID NO:7;

[0020] And the LCDR3 is shown in SEQ ID NO:8.

[0021] As used herein, the terms "variable region" and "CDR" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to the regions containing one or more or even all of the major amino acid residues that contribute to the binding affinity of the antibody or antigen-binding fragment to its recognized antigen or epitope.

[0022] Herein, the heavy chain variable region (heavy chain complementary determining region) is represented by "HCDR", which includes HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by "LCDR", which includes LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. 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 KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The present disclosure uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods also fall within the scope of protection of the present invention.

[0023] Herein, "framework region" or "FR" region includes heavy chain framework region and light chain framework region, and refers to the region excluding CDR in antibody heavy chain variable region (which can be expressed as VH) and light chain variable region (which can be expressed as VL); wherein, the heavy chain framework region is expressed as "HFR" and can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region is expressed as "LFR" and can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0024] Preferably, the antibody or functional fragment thereof 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,

[0025] 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;

[0026] 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;

[0027] 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;

[0028] 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;

[0029] 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;

[0030] 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;

[0031] 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;

[0032] 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.

[0033] In this article, the heavy chain variable region is obtained by arranging and connecting the CDRs and FRs with the following numbers in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the CDRs and FRs with the following numbers in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0034] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the antibodies or their functional fragments provided by the present 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 above-mentioned corresponding framework regions (SEQ ID NO: 9, 10, 11, 12, 13, 14, 15 or 16). In an optional embodiment, the antibody or its functional fragment has a KD of ≤10 -7 M, KD≤10 -8 M, KD≤10 -9 M, KD≤10 -10 M or KD≤10 -11 Binds human IgM with an affinity of .

[0035] Preferably, the antibody or its functional fragment further comprises a constant region;

[0036] Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region;

[0037] Optionally, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region;

[0038] 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;

[0039] Optionally, the species origin of the constant region is mouse.

[0040] Preferably, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0041] The functional fragments of the above antibodies generally have the same binding specificity as the antibodies from which they are derived. It is easy for a person skilled in the art to understand based on the contents of the disclosure that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the disclosure, a person skilled in the art can easily obtain the above functional fragments.

[0042] On the other hand, the present invention discloses an antibody conjugate, which comprises the antibody or its functional fragment and a conjugated part conjugated thereto;

[0043] Optionally, the antibody or functional fragment thereof is labeled with a marker;

[0044] Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels;

[0045] 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;

[0046] Optionally, the enzyme is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and 6-phosphoglucose deoxygenase;

[0047] 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;

[0048] 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;

[0049] Optionally, the nanoparticle-based marker is selected from nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles;

[0050] Optionally, the colloid is selected from colloidal metals, disperse dyes, dye-labeled microspheres and latex;

[0051] Optionally, the colloidal metal is selected from colloidal gold, colloidal silver and colloidal selenium;

[0052] Optionally, the antibody or functional fragment thereof is coated onto a solid phase;

[0053] Optionally, the solid phase is selected from microspheres, plates and membranes;

[0054] Optionally, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0055] On the other hand, the present invention discloses a detection reagent or a kit, wherein the reagent or the kit comprises the antibody or a functional fragment thereof or the antibody conjugate.

[0056] In an optional embodiment, the blocking agent included in the kit may be in the form of a liquid solution, in the form attached to a solid support, or in the form of a dry powder. When the blocking agent is a liquid solution, the liquid solution may be an aqueous solution. When the immunoblocking agent is in the form attached to a solid support, the preferred solid support may be a chromatographic medium such as a film, a test strip, a plastic bead or a plate, or a microscope slide. When the blocking agent is a dry powder, the powder may be reconstituted by adding an appropriate solvent.

[0057] In another aspect, the present invention discloses a nucleic acid encoding the antibody or a functional fragment thereof.

[0058] Nucleic acids are typically RNA or DNA, and nucleic acid molecules can be single-stranded or double-stranded. A nucleic acid is "operably linked" when it is 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 operably linked to the coding sequence. DNA nucleic acids are used when they are linked to a vector.

[0059] On the other hand, the present invention also provides a vector containing the above nucleic acid molecule.

[0060] In another aspect, the present invention also provides a cell containing the above-mentioned vector.

[0061] On the other hand, the present invention also provides a method for preparing an antibody or a functional fragment thereof, which comprises: culturing the cells as described above, and separating and purifying the antibody or the functional fragment thereof from the culture product.

[0062] Based on the amino acid sequence of the antibody or its functional fragment in the present disclosure, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment, for example, separating and purifying the antibody or its functional fragment from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment in the present disclosure, it belongs to the protection scope of the present disclosure.

[0063] 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 a progesterone protein detection product.

[0064] In another aspect, the present invention discloses a method for detecting progesterone protein in a sample, characterized in that the method comprises:

[0065] a) contacting the progesterone protein antigen in the test sample with the antibody or its functional fragment, the antibody conjugate, or the reagent or kit under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and

[0066] b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample;

[0067] Optionally, in step a), the immune complex further comprises a second antibody, which binds to the antibody or antigen-binding fragment;

[0068] Optionally, in step a), the immune complex further comprises a second antibody, and the second antibody binds to the progesterone protein antigen.

[0069] Beneficial Effects

[0070] The antibody sequence disclosed in the present invention has stable antibody performance and high affinity for progesterone small molecules. The human progesterone detection chemiluminescent diagnostic reagent developed based on the antibody shows excellent performance in progesterone detection in clinical tests and has important clinical practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 : The SDS-PAGE electrophoresis test results of the antibody disclosed in the present invention;

[0072] Figure 2 : The test results of the reactivity between the antibody disclosed in the present invention and progesterone;

[0073] Figure 3 :The results of chemiluminescence reagent detection of progesterone content in clinical samples. DETAILED DESCRIPTION

[0074] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0076] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0077] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0078] Example 1 Preparation of progesterone immunogen

[0079] Long-arm progesterone (progesterone-11alpha-carboxymethylether) is obtained by chemical synthesis by dissolving in DMSO, and activated by EDC (N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to obtain the active intermediate progesterone-NHS, which is then coupled with KLH (hemocyanin) or OVA (ovalbumin) to obtain progesterone-KLH or progesterone-OVA conjugates. After the coupling is completed, the unbound small molecules are removed by dialysis in phosphate buffer PBS to obtain a progesterone conjugate that can be used for immune or antibody activity analysis.

[0080] Example 2 Animal Immunization

[0081] Three healthy female sheep aged 6-12 months were selected for immunization. For the first immunization, 500ug of progesterone-KLH immunogen was used, mixed with Freund's complete adjuvant in a 1:1 ratio and emulsified, and immunized at multiple points such as the left / right armpit, around the cervical lymph nodes, and on the back of the shoulder. After that, booster immunizations were performed at intervals of 3 weeks using Freund's incomplete adjuvant. After each immunization, serum was collected for titer testing, and the serum titer was qualified when it was >1 million (6-9 immunizations). One week after the last immunization, peripheral blood of the sheep was collected and PBMC was isolated.

[0082] Example 3 Preparation of peripheral blood PBMC

[0083] Select immune sheep with higher titers, collect 50mL of fresh blood, and dilute the blood sample with PBS 1:1. Take 5 50mL centrifuge tubes, add 20mL of Histopaque (Sigma) separation solution to each tube, carefully add 20mL of diluted blood sample to the surface of the Histopaque separation solution in each tube, and centrifuge at 400g for 35 minutes at room temperature to separate PBMC. After centrifugation, granulocytes, platelets, and red blood cells will pass through the separation solution and be located at the bottom of the centrifuge tube, while PBMC will be above the Histopaque separation solution. The collected PBMCs are washed twice with PBS and centrifuged at 300g to collect the cells.

[0084] Example 4 Phage display library construction

[0085] Use freshly collected sheep PBMC cells and add 1 mL TRIzol per 1E7 cells. TMReagent. Mix by inversion repeatedly until all cells are dissolved and there is no obvious precipitation, and leave at room temperature for 10 minutes. Add 1 / 5 volume of chloroform to the solution 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, mix thoroughly by inversion, and let stand at room temperature for 30 minutes to precipitate RNA. Collect the RNA precipitate by centrifugation at 12000g for 10 minutes at 4°C, wash the RNA precipitate with 800ul of 75% ethanol, evaporate the ethanol, dissolve the RNA with 50ul of RNase-free water and quantify. Use PrimeScript TM II Reverse Transcriptase (2690A) was used to prepare cDNA by reverse transcription according to the instructions. A sheep scFv immune library against progesterone was constructed according to the published experimental method. (J Immunol Methods. 2000 Mar 6; 236 (1-2): 133-46.;; Phage Display—A Practical Approach. Edited by T.Clackson and HBLowman Oxford University Press, Oxford, UK; 2004;)

[0086] The PCR primers for cloning sheep V region genes used in the present invention are shown in SEQ ID NOs: 17-37, 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 NOs: 16-36, respectively.

[0087] Phanta Max Super-Fidelity DNA Polymerase (P505-d3) produced by Nanjing Novozymes Biotechnology Co., Ltd. was used for amplification of each gene fragment and splicing and assembly of scFv.

[0088] PCR amplification conditions were as follows: 95°C pre-denaturation for 3 min (1 cycle); 95°C denaturation for 13 s, 56°C annealing for 15 s, 72°C extension for 30 s (30 cycles); 72°C final extension for 5 min (1 cycle). Each amplified fragment was purified and recovered using a DNA agarose gel purification kit (OMEGA, D2500-02). The spliced ​​scFv fragment needed to be digested with SfiI restriction endonuclease (NEB, R0123L) and then connected to the pComb3X phagemid 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, the transformed TG1 cells were all plated on a plate containing 100 ug / mL Amp antibiotic and cultured overnight. The resulting culture was the gene library. The TG1 gene library was infected with M13K07 helper phage (NEB, N0315S) and then cultured overnight. The culture supernatant was precipitated with polyethylene glycol to prepare a phage display library.

[0089] Example 5 Phage-antibody panning and clone identification

[0090] In order to obtain the appropriate antibodies described in the present invention by panning, the antigens progesterone-OVA and progesterone-KLH were diluted to 100ug / ml, 10ug / ml, 1ug / ml and 0.1ug / ml respectively using PBS and coated on immunotubes (NUNC MaxiSorp TM). 5E12 phage-scFv particles were incubated with the coated antigen and washed with PBS solution containing 0.1% Tween 20. The concentration of the coated antigen was gradually reduced in each round of panning, and the number of washes was increased. The bound target phage was eluted with 10mM HCl and the elution solution was neutralized with 1M Tris-HCl, pH8.3. The eluted target phage was infected with fresh exponentially proliferating TG1 cells and plated on YTAG plates (1.6% tryptone, 1% yeast extract, 0.5% sodium chloride, 100ug / mL ampicillin, 2% glucose) to obtain single clones. A single clone was picked and cultured to OD600 of 0.5, infected with M13K07 helper phage (NEB, N0315S), and cultured overnight in YTAK medium (1.6% tryptone, 1% yeast extract, 0.5% sodium chloride, 100ug / mL ampicillin, 50ug / mL kanamycin). A large number of phage-scFv monoclonal particles were present in the culture supernatant. The culture supernatant was used for ELISA to identify phage clones that specifically bind to progesterone-OVA and progesterone-KLH. In the monoclonal identification, phage clones with strong binding ability to progesterone-OVA and progesterone-KLH will be tested again for clones in which free progesterone small molecules have a significant inhibitory effect on the binding, and the results are shown in Table 1. The phage clone finally selected (clone-3 in Table 1) was sequenced by conventional genes to obtain the gene coding sequence of the phage-scFv.

[0091] Table 1. ELISA identification of the reactivity of the obtained phage monoclones to progesterone.

[0092]

[0093] Example 6 Recombinant expression and purification of sheep monoclonal antibodies

[0094] The complete scFv sequence of clone-3 was sequenced, and the VH and VL sequences of the corresponding sheep monoclonal antibody were analyzed. The complete antibody heavy chain and antibody light chain sequences were constructed into the pcDNA 3.1 vector (Thermo V79020) suitable for eukaryotic expression. The prepared endotoxin-free antibody expression plasmids were mixed in a ratio of heavy chain: light chain = 1:1 and then used Lipofectin TM Transfection reagent (Thermo) was used to transfect HEK293 cells. After 5 days of culture, the culture supernatant was collected by centrifugation. The culture supernatant contained the expressed sheep monoclonal antibody, which was purified by protein G affinity to obtain the target antibody. The SDS electrophoresis monitoring results of the purified anti-progesterone sheep monoclonal antibody are shown in Figure 2. Figure 1. As shown. Among them, the reduction treatment of the antibody is to take 20ug of antibody, add 10ul of reduced protein loading buffer (Biyuntian P0015L), dilute to a total volume of 50ul with pure water, and heat at 100℃ for 10min to fully denature the antibody protein. The non-reduction treatment of the antibody is to take 20ug of antibody, add 10ul of non-reduced protein loading buffer (Biyuntian P0016N), dilute to a total volume of 50ul with pure water, mix well and directly spot.

[0095] Example 7 Monitoring the Reactivity of Anti-Progesterone Antibodies to Progesterone

[0096] The prepared progesterone-KLH was diluted with coating solution (0.05M pH = 9.5 carbonate buffer) at 1ug / mL, and 100μL / well was added to the ELISA plate and coated overnight at 4°C. During the detection, the antibody to be tested was diluted to the target concentration (from 1000ng / mL to 1.4ng / mL) and incubated with the antigen at 37°C for 1h, and HRP-labeled rabbit anti-sheep polyclonal antibody was used for color development, and the absorbance value of OD450 was detected. The results are shown in Table 2 below. Figure 2 To further determine the binding ability of the antibody to natural progesterone, the antibody was diluted to 100 ng / mL and incubated with different concentrations of free progesterone molecules at 37°C for 30 minutes before being added to the ELISA wells coated with progesterone-KLH. Free progesterone will compete with the antibody for its binding ability to progesterone-KLH. The test results are shown in Table 3.

[0097] Table 2. Results of monitoring the reactivity of anti-progesterone antibodies with progesterone

[0098] Antibody concentration (ng / mL) OD450 1000 1.895 333.3 1.317 111.1 0.761 37.1 0.372 12.3 0.174 4.1 0.11 1.4 0.062 0 0.045

[0099] Table 3. Competitive binding of free progesterone small molecules to progesterone antibodies.

[0100]

[0101]

[0102] Example 8 Anti-progesterone antibody used as chemiluminescence (competitive method) detection reagent

[0103] Use EZ-Link according to the public method TMNHS-LC-LC-biotin (thermo, 21343) was used to biotin-label the anti-progesterone antibody, and a desalting column was used to remove free unbound biotin. The biotin-labeled antibody was diluted to 0.1ug / mL using a specific buffer to obtain the R1 reagent. Alkaline phosphatase-coupled progesterone was prepared with a similar scheme as in Example 1, and diluted to 1.2ug / ml using a specific buffer to obtain the R2 reagent. The chemiluminescence detection of clinical samples was performed using the EXI1800 fully automatic chemiluminescence immunoassay analyzer independently developed by our company. After each clinical sample was mixed with the R1 reagent, it was incubated with streptavidin-coupled magnetic beads for reaction, and then the R2 reagent was added and mixed for reaction. The detection was performed according to the operating instructions of the EXI1800 fully automatic chemiluminescence immunoassay analyzer, and the test results are shown in Tables 4 and Figure 3 Judging from the test results, the antibody of the present invention has excellent clinical detection performance.

[0104] Table 4. Results of chemiluminescence-based progesterone content detection in clinical samples using the antibodies of the present invention.

[0105]

[0106]

[0107] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. An anti-human progesterone antibody or a functional fragment thereof, characterized in that: It comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1 to HCDR3 are amino acid sequences consistent with HCDR1 to HCDR3 of the heavy chain variable region shown in SEQ ID NO:1, and the LCDR1 to LCDR3 are amino acid sequences consistent with LCDR1 to 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.

5. The antibody or functional fragment thereof according to any one of claims 1 to 4, characterized in that: 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, IgM, 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; Optionally, the species origin of the constant region is mouse. Preferably, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

6. 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.

7. 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 6.

8. A nucleic acid, characterized in that The antibody or its functional fragment encoded by any one of claims 1 to 5 9. Use of the antibody or functional fragment thereof according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 in the preparation of a progesterone protein detection product.

10. A method for detecting progesterone protein in a sample, characterized in that: The method comprises: a) contacting the progesterone 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 6, or the reagent or kit according to claim 7 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 progesterone protein antigen.

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