Prolactin antibody or functional fragment thereof and application thereof

Abstract

The invention discloses a prolactin antibody and application thereof, and relates to the field of antibodies. The prolactin antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for detection of prolactin, and has good affinity or activity.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically to an antibody against prolactin and its applications. Background Technology

[0002] Prolactin (PRL), also known as lactogenic hormone, is a hormone secreted by the anterior pituitary gland and plays a crucial role in the initiation and maintenance of lactation. Measuring prolactin levels not only examines the pituitary's prolactin reserve but also helps diagnose pituitary diseases and has significant clinical value in the diagnosis and differential diagnosis of menstrual abnormalities and infertility. PRL is synthesized and intermittently secreted by the anterior pituitary gland, with the mammary gland as its target organ, responsible for its maturation and differentiation. High concentrations of PRL inhibit steroid production in the ovary and the production and secretion of pituitary gonadotropins. It is used in research on pituitary microtumors, reproductive biology, and drug-induced hyperlactation.

[0003] PRL is a polypeptide containing 199 amino acids and three disulfide bonds, with a molecular weight of 22,000. Larger PRL molecules also exist in the blood, possibly as PRL precursors or aggregates of several PRLα molecules. In adult males, the plasma PRL concentration is <20 μg / L. In women, the PRL level in the body varies cyclically: <23 ug / ml during the follicular phase, between 5-40 ug / L during the luteal phase, and gradually increases to over 400 ug / L as pregnancy progresses.

[0004] There are many methods for detecting PRL, primarily based on the antigen-antibody sandwich method. This method offers advantages such as low cost, simple operation, and suitability for large-scale screening, making it the most commonly used detection method currently. In clinical testing, chemiluminescent immunoassay and immunochromatographic assays are the mainstream methodologies, both of which are optimizations and upgrades of the original double-antibody sandwich method. These methods mostly rely on the specific binding reaction between antigen and antibody and require antibodies targeting PRL. Therefore, there is a strong demand in the field of PRL detection for high-performance anti-PRL antibodies. Specific Implementation

[0005] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0006] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0007] 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. The methods and materials described herein, and any similar or equivalent methods and materials, may be used in the practice or testing of formulations or unit doses described herein. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0008] As used herein, the terms “comprising,” “including,” “having,” “may,” and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures.

[0009] In a first aspect, embodiments of the present invention provide an antibody or antigen-binding fragment thereof against prolactin, said antibody or antigen-binding fragment thereof comprising a complementarity-determining region of any one of the following groups:

[0010] (1) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18 respectively;

[0011] (2) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:21, SEQ ID NO:16, SEQ ID NO:23 and SEQ ID NO:18 respectively;

[0012] (3) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:16, SEQ ID NO:23 and SEQ ID NO:24 respectively;

[0013] (4) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:19, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:22, SEQ ID NO:17 and SEQ ID NO:18 respectively;

[0014] (5) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:15, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:18 respectively;

[0015] (6) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24 respectively.

[0016] It should be noted that HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to those of HCDR1, HCDR2, and HCDR3 in the same heavy chain variable region defined in the antibody or its antigen-binding fragment described in the first aspect, and LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to those of LCDR1, LCDR2, and LCDR3 in the same light chain variable region defined in the antibody or its antigen-binding fragment described in the first aspect.

[0017] In this invention, the term "antibody" is used in the broadest sense, and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, as long as they exhibit the desired biological activity.

[0018] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0019] In this invention, the heavy chain complementarity determination region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determination region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0020] The methods for defining CDRs are well-known in the art and include: the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As described herein, the “Kabat definition” refers to the definition system described by Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). For the “Chothia definition,” see Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may shorten or lengthen them based on predictions or experimental results for specific residues or residue groups.

[0021] According to an embodiment of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact, preferably defined by the Kabat system.

[0022] In this invention, the "frame region" or "FR" region includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0023] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination:

[0024] LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0025] In some specific embodiments, the antibody or its antigen-binding fragment described in this invention further comprises frame regions HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4;

[0026] The HFR1 includes SEQ ID NO:3 or an amino acid sequence that has at least 80% identity with it;

[0027] The HFR2 comprises SEQ ID NO:4 or an amino acid sequence having at least 80% identity with it;

[0028] The HFR3 includes SEQ ID NO:5 or an amino acid sequence that is at least 80% identical to it;

[0029] The HFR4 comprises SEQ ID NO:6 or an amino acid sequence having at least 80% identity with it;

[0030] The LFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it;

[0031] The LFR2 comprises SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it;

[0032] The LFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity with it;

[0033] The LFR4 includes SEQ ID NO:10 or an amino acid sequence that is at least 80% identical to it.

[0034] It should be noted that, in other embodiments, the amino acid sequences of each frame region of the prolactin antibody or its antigen-binding fragment provided by the present invention 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% identity with the corresponding frame regions (SEQ ID NO: XX) described above.

[0035] Secondly, embodiments of the present invention provide an antibody against prolactin or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO:1, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:33; and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:34.

[0036] In an optional embodiment, the antibody or antigen-binding fragment thereof described in the first and second aspects above further includes a constant region.

[0037] In an optional implementation, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0038] In an optional implementation, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0039] In an optional embodiment, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0040] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0041] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0042] In an optional implementation, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

[0043] In an optional implementation, the species source of the constant region is mice.

[0044] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:11, and the light chain constant region sequence (CL) is as shown in SEQ ID NO:12.

[0045] It should be noted that, in other embodiments, the constant region sequence 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% identity with the aforementioned constant regions (SEQ ID NO: 11-12).

[0046] In an optional embodiment, the antigen-binding fragment is selected from any one of the antibody's F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv.

[0047] The antigen-binding fragments of the aforementioned antibodies typically possess the same binding specificity as the source antibody. Those skilled in the art will readily understand, based on the description of this invention, that the antigen-binding 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 invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0048] The antigen-binding fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0049] Thirdly, the present invention provides an antibody conjugate comprising the antibody or its antigen-binding fragment described above.

[0050] In an optional embodiment, the antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody or its antigen-binding fragment.

[0051] In an optional embodiment, the antibody conjugate further includes a marker conjugated to the antibody or its antigen-binding fragment.

[0052] In an optional implementation, the aforementioned marker refers to a type of substance that has properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. Through these properties, qualitative or quantitative detection of the corresponding target can be achieved.

[0053] In optional embodiments, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0054] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0055] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0056] In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0057] In optional embodiments, the radioactive isotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0058] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0059] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0060] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0061] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0062] In an optional embodiment, the colloidal metal is colloidal gold.

[0063] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

[0064] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0065] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0066] Fourthly, the present invention provides a reagent or kit comprising the above-described antibody or its antigen-binding fragment or the above-described antibody conjugate.

[0067] As previously stated, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention can effectively bind to prolactin. Therefore, reagents or kits containing the prolactin antibody or antigen-binding fragment can effectively perform qualitative or quantitative detection of prolactin. The reagents or kits provided by the present invention can be used, for example, in immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, latex immunoturbidimetry, and other detection methods involving the specific binding properties of prolactin and its antibodies. As previously stated, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention have higher binding activity or affinity for prolactin; therefore, reagents or kits containing the antibody or antigen-binding fragment have higher detection sensitivity or specificity.

[0068] Fifthly, the present invention provides a method for detecting prolactin, comprising: a) contacting the antibody or its antigen-binding fragment, antibody conjugate, reagent or kit with prolactin in a sample to be tested under conditions sufficient to induce 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;

[0069] In an optional embodiment, the immune complex further includes a second antibody that binds to the antibody or its antigen-binding fragment.

[0070] In an optional embodiment, the immune complex further includes a second antibody that binds to prolactin.

[0071] In a sixth aspect, the present invention provides the use of the above-described prolactin antibody or its antigen-binding fragment, antibody conjugate, or the above-described reagent or kit in the preparation of products for detecting prolactin.

[0072] In a seventh aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or its antigen-binding fragment.

[0073] Eighthly, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0074] In a ninth aspect, the present invention provides cells containing the above-described carrier.

[0075] In a tenth aspect, the present invention provides a method for preparing prolactin antibodies or antigen-binding fragments thereof, comprising: culturing cells as described above.

[0076] Based on the amino acid sequence of the prolactin antibody or its antigen-binding fragment disclosed in this invention, those skilled in the art will readily conceive of preparing the prolactin antibody or its antigen-binding fragment using genetic engineering or other techniques (chemical synthesis, recombinant expression). For example, the antibody or its antigen-binding fragment can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody or its antigen-binding 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 prolactin antibody or its antigen-binding fragment of this invention, it falls within the protection scope of this invention.

[0077] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the 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.

[0078] 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.

[0079] Unless otherwise specified, the practice of this invention 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); *Oligo Nucleotide 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., 1991), each of which is explicitly incorporated herein by reference.

[0080] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0081] Example 1: Preparation of prolactin antibodies

[0082] (1) Mouse immunization and antibody detection

[0083] Five 6-8 week old SPF-grade female BALB / c mice were selected. Freund's complete adjuvant and PRL protein (purchased from LEEbio) at a concentration of 1 mg / ml were mixed in equal volumes and emulsified. The emulsified antigen was used to immunize the 6-8 week old SPF-grade female BALB / c mice, with each mouse receiving 50 μg of antigen protein via paw injection. Two weeks after the initial immunization, the antigen protein was mixed with Freund's incomplete adjuvant and emulsified, and each mouse received 40 μg of antigen protein via paw injection or subcutaneous injection in the back. Two weeks later, blood was collected via tail vein, the supernatant was collected by centrifugation, and serum titer was determined by ELISA. Immunization was repeated every two weeks, and serum titer was measured. After two immunizations, the serum titer, after a million-fold dilution, was higher than 2.0. A serum titer of 10 was selected for screening. 6 Lymphocytes were isolated from the mice mentioned above for cell fusion.

[0084] (3) Hybridoma antibody screening

[0085] The antibody screening protocol is crucial for the successful development of the antibodies of this invention. In the antibody screening stage, the ELISA plate is first coated with PRL protein. After the potential target antibody binds to the PRL protein, it is then developed using goat anti-mouse HRP and ELISA reagent to screen for highly reactive hybridoma clones. Secondly, the ELISA plate is coated with TSH / FSH / hCG protein (purchased from LEEbio). If the potential target antibody binds to the TSH / FSH / hCG protein, and then the ELISA reagent is developed using goat anti-mouse HRP, it indicates that the antibody will be interfered with by these proteins and should be excluded.

[0086] The target clones obtained through screening need to meet the following conditions: 1. Good reactivity to PRL protein; 2. No significant reactivity to TSH / FSH / hCG protein. The PRL-A cell line screened in this invention was used for antibody A preparation.

[0087] (4) Production and purification of monoclonal antibodies

[0088] Two groups of 6-8 week old BALB / c mice were selected, and 500 μL of paraffin oil was injected intraperitoneally to suppress the immune response. One week after injection, 0.5 ml of PRL-A hybridoma cells (approximately 1 × 10⁻⁶ cells) were injected intraperitoneally into one group of mice. 6 Quantity. Ascites fluid collection began two weeks later. The collected ascites fluid was purified by ammonium sulfate precipitation and protein G affinity purification to obtain the target antibody.

[0089] (5) Identification of monoclonal antibody subtypes and cloning of gene sequences

[0090] The SBA Clonotyping System-HRP kit from Southern Biothech was used to identify the heavy and light chain isotypes of monoclonal antibodies, following the manufacturer's instructions. The specific procedure was as follows:

[0091] a. Dilute the capture antibody to 1 μg / mL with coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), add 100 μL / well to the microplate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20.

[0092] b. Dilute the culture supernatant of the hybridoma cells to be tested 1:1 with diluent (1% BSA, 0.1% PBST), add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 min. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lambda-HRP) 1:3000 with diluent.

[0093] After washing the plate three times with washing buffer, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing three more times, add chromogenic buffer and incubate for approximately 5 minutes (depending on the reaction strength), then add 2M sulfuric acid to terminate the reaction and read the OD450 absorbance. Identification revealed that the heavy chain isotype of the PRL-A antibody is IgG1, and the light chain is Kappa. Based on the antibody isotype results, the antibody gene sequence was cloned using a RACE-based method. Hybridoma cells in good growth condition were collected, and total RNA was obtained from the hybridoma cells using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the Takara SMARTer RACE instructions, and the full-length sequence of the target antibody was amplified.

[0094] The obtained antibody was named PRL-A. PRL-A was mutated, and five mutant antibodies with equivalent performance were screened out. The antibody sequences are shown in Table 1 below.

[0095] Table 1 Antibody Sequences

[0096]

[0097]

[0098] *The numbers for all CDR definitions in the table are based on the Kabat numbering system.

[0099] Example 2: Antibody Performance Detection

[0100] 1. Reactivity identification

[0101] PRL antigen was diluted to 1 μg / mL with 50 mM carbonate buffer and coated onto microplates at 100 μL per well, incubated overnight at 4°C. The next day, the plates were washed twice with PBST and patted dry. The above antibodies were added, serially diluted 3-fold starting from 1000 ng / mL, and loaded at 100 μL / well, incubated at 37°C for 30 min (with some supernatant added for 1 h). The plates were washed 5 times with PBST and patted dry. Horseradish peroxidase-labeled goat anti-mouse IgG was added at 100 μL / well, incubated at 37°C for 30 min, washed 5 times with PBST and patted dry. Urea peroxide (50 μL / well) and tetramethylbenzidine (50 μL / well) were added, and the reaction was incubated for 10 min. The reaction was stopped by adding dilute hydrochloric acid at 50 μL / well. The OD values ​​were read at 450 nm (reference 630 nm) on a microplate reader. The antibody reactivity was identified as shown in Table 2 below.

[0102] Table 2 Reactivity data

[0103]

[0104] The experimental results show that all six prolactin antibodies exhibited good reactivity.

[0105] 2. Functional testing

[0106] A. Biotin-labeled antibody PRL

[0107] Weigh out biotin (NHS-LC-LC-Biotin, from Thermo Scientific, 21343), dissolve it in DMSO to a concentration of 5.677 mg / mL, take 0.3 mg of PRL (self-produced) for a total of 6 antibody strains, the required volume of biotin solution for each 1 mg antibody is 3.3 μL, add biotin and mix well, ligate the antibody to biotin, and react at 25°C in the dark for 4 h.

[0108] B. Alkaline phosphatase-labeled antibody

[0109] Displace 0.3 mg of PRLA-F antibody into TSE (pH 8.5) buffer, and displace 0.66 mg of ALP (from Sigma, ALPI12G) into ALP dialysis buffer (pH 7.6) solution. Mix well and determine the concentration. Dissolve the weighed Traut's Reagent 2-Iminothiolane·HCl (abbreviated as 2-IT, from Thermo Scientific, 26101) in TSE (pH 8.5) solution to a concentration of 13.76 mg / mL. Dissolve the weighed Sulfo-SMCC (from Thermo Scientific, PG82085) in purified water to a concentration of 3.7 mg / mL. Both activator solutions should be prepared fresh and used within 10 minutes of preparation. Calculate the required volume of 2-IT solution for this step based on a requirement of 5 μL per 1 mg of antibody. Add the 2-IT solution to the antibody, mix well, and allow to stand at (25±2) °C for 20 min. This step is antibody activation. After activation, the activated antibody was transferred to TSE (pH 7.3) solution, and the antibody concentration was measured. Simultaneously, the required volume of Sulfo-SMCC solution was calculated based on 10 μL of SMCC solution per 1 mg of ALP. The Sulfo-SMCC solution was added to the desalted ALP, mixed thoroughly, and allowed to stand at (25±2) °C for 20 min. After activation, the activated ALP was transferred to TSMZ (pH 7.3) solution, and the ALP concentration was measured. The desalted antibody was diluted to 0.3 mg / mL with TSE (pH 7.3) solution, and the desalted ALP was diluted to 0.4 mg / mL with TSMZ (pH 7.3) solution. The antibody and ALP volumes for ligation were calculated based on a 1:0.91 mass ratio. The calculated antibody and ALP volumes were mixed and allowed to stand at 2–8 °C for 12–20 h. Dissolve maleimide in DMSO to a concentration of 9.7 mg / mL, and dilute 10-fold with TSMZ (pH 7.3) to obtain the stop solution. Add 20 μL of the stop solution required for 1 mL of the antibody-ALP conjugate to terminate the reaction.

[0110] C. Performance Evaluation

[0111] (1) Linear Experiment

[0112] The assay reagents include R1, R2, and streptavidin magnetic bead solution:

[0113] R1 comprises: a biotin-labeled PRL antibody (the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:35, and the light chain amino acid sequence is shown in SEQ ID NO:36), with a concentration of 1 μg / mL.

[0114] R2 comprises: alkaline phosphatase-labeled PRL-A / B / C / D / E / F antibody, used at a concentration of 1 μg / mL.

[0115] The specific experimental procedures are as follows:

[0116] a. Experimental preparation: 5 mL each of R1 and R2 working solutions, 4 mL of magnetic bead solution, and WRSA-WRS F calibrators.

[0117] b. 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 the anti-PRL item (30uL sample, 145uL R1, 245uL R2, 30uL magnetic bead solution) for the experiment.

[0118] Table 3. Results of the linear experiment

[0119] WRS PRL-A PRL-B PRL-C PRL-D PRL-E PRL-F WRS-A 5549 5275 5412 7025 6458 6741.5 WRS-B 580356 621092 600724 539233 555034 547133.5 WRS-C 2738541 2794073 2766307 2583359 2571985 2577672 WRS-D 9825650 10552352 10189001 9254462 9554389 9404425.5 WRS-E 35982477 35913762 35948119.5 32727065 32865900 32796482.5 WRS-F 62093599 62215976 62154787.5 60281905 58111027 59196466

[0120] Therefore, as shown in Table 3, the above six anti-prolactin antibodies, when used as labeling antibodies in PRL chemiluminescence detection, all exhibited good linearity.

[0121] 3. Clinical trials

[0122] The specific experimental procedures are as follows:

[0123] (1) Experimental preparation: Take out 20 PRL samples, thaw them at room temperature, and mix them thoroughly on a vortex mixer;

[0124] (2) Testing: Antibodies against PRL-A and the mutant PRL-C were extracted from 20 clinical samples and tested respectively. The clinical results are shown in Table 4 below:

[0125] Table 4 Clinical Trial Results

[0126]

[0127]

[0128] The clinical data above show that the clinical values ​​of PRL-A and the mutant strain PRL-C meet the requirements and can be used in in vitro diagnostic reagents.

[0129] All data, reagents, and procedures described herein should be understood as illustrative rather than restrictive. Although the invention has been described in conjunction with the specific embodiments described above, many modifications and other variations will be apparent to those skilled in the art. All such modifications and other variations also fall within the scope of the invention.

Claims

1. An antibody against prolactin or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment includes a complementarity-determining region from any of the following groups: (1) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18 respectively; (2) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:21, SEQ ID NO:16, SEQ ID NO:23 and SEQ ID NO:18 respectively; (3) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:16, SEQ ID NO:23 and SEQ ID NO:24 respectively; (4) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:19, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:22, SEQ ID NO:17 and SEQ ID NO:18 respectively; (5) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:15, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:18 respectively; (6) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24 respectively.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The complementary determination region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact, preferably by the Kabat system.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further includes the framework regions HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; The HFR1 includes SEQ ID NO:3 or an amino acid sequence that has at least 80% identity with it; The HFR2 comprises SEQ ID NO:4 or an amino acid sequence having at least 80% identity with it; The HFR3 includes SEQ ID NO:5 or an amino acid sequence that is at least 80% identical to it; The HFR4 comprises SEQ ID NO:6 or an amino acid sequence having at least 80% identity with it; The LFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it; The LFR2 comprises SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it; The LFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity with it; The LFR4 includes SEQ ID NO:10 or an amino acid sequence that is at least 80% identical to it.

4. An antibody against prolactin or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO:1, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:33; and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:

34.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region; Preferably, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Preferably, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans; Preferably, the heavy chain constant region includes an amino acid sequence of SEQ ID NO:11 or having at least 80% identity with it, and the light chain constant region includes an amino acid sequence of SEQ ID NO:12 or having at least 80% identity with it.

6. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5; Preferably, the antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody or its antigen-binding fragment; Preferably, the antibody conjugate further includes a marker conjugated to the antibody or its antigen-binding fragment; Preferably, the marker is selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers; Preferably, the antibody conjugate further includes a solid-phase carrier conjugated to the antibody or its antigen-binding fragment; Preferably, the solid support is selected from at least one of microspheres, plates, or membranes; Preferably, the solid support is selected from at least one of magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon or nitrocellulose membranes.

7. A reagent or kit, characterized in that, The reagent or kit comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5 or the antibody conjugate as described in claim 6; Preferably, the method is selected from at least one of immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, and latex immunoturbidimetry.

8. A method for detecting prolactin, characterized in that, include: a) Under conditions sufficient to induce an antibody / antigen binding reaction, the antibody or antigen-binding fragment thereof of any one of claims 1-5, the antibody conjugate of claim 6, or the reagent or kit of claim 7 are brought into contact with prolactin in the sample to be tested to form an immune complex. and b) Detect the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample; Preferably, the immune complex further includes a second antibody, which binds to the antibody or its antigen-binding fragment; Preferably, the immune complex further includes a second antibody that binds to prolactin.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 in the preparation of products for detecting prolactin.

10. A nucleic acid, a vector, a cell, or a method for preparing an antibody or an antigen-binding fragment thereof according to any one of claims 1-5, wherein the nucleic acid encodes an antibody or an antigen-binding fragment thereof according to any one of claims 1-5; the vector contains a nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-5; the cell contains the aforementioned nucleic acid or vector; and the method comprises the aforementioned cell.

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