Method for rapidly predicting operation effect in hyperparathyroidism operation

Abstract

The invention discloses a method for rapidly predicting an operation effect in a hyperparathyroidism operation, and belongs to the technical field of medical diagnosis. The invention develops a detection product and designs a novel method for rapidly predicting the surgical effect in hyperparathyroidism surgery, which comprises the following steps: respectively collecting blood samples before and after hyperparathyroidism surgery, and detecting the content of parathyroid hormone in the blood samples by adopting the detection product, obtaining an operation effect prediction result according to the change of the parathyroid hormone content before and after the operation; the detection product contains an anti-parathyroid hormone antibody secreted by a hybridoma cell strain with the preservation number of CCTCC (China Center For Type Culture Collection) No.2025122. According to the invention, the defects of long detection time and harsh sample requirements in the prior art are overcome, the change of the PTH content in the blood sample can be rapidly and accurately detected, whether hyperparathyroid gland excision is complete or not can be judged in an auxiliary manner, the detection time is shortened to 20 minutes, and the operation time is greatly shortened.

Description

Technical Field

[0001] The present invention relates to a method for rapidly predicting surgical effects in hyperparathyroidism surgery, belonging to the technical field of medical diagnosis. Background Art

[0002] Hyperparathyroidism is a common endocrine disease, and the most effective treatment is to remove the hyperfunctioning parathyroid glands. The current conventional method for determining the effectiveness of hyperparathyroidism surgery is to identify the hyperfunctioning parathyroid glands based on preoperative imaging (ultrasound, CT, MIBI) and then remove them. However, due to the insensitivity of some parathyroid glands on imaging and the problem of ectopic parathyroid glands, it is impossible to visualize all diseased parathyroid glands before surgery. Although the parathyroid glands located on imaging are removed during surgery, other diseased parathyroid glands are missed, resulting in no improvement in hyperparathyroidism and surgical failure. Therefore, a new method is needed to guide the surgical procedure. Summary of the Invention

[0003] To solve the above problems, the present invention provides a new method that overcomes the defect of long serum parathyroid hormone detection time in existing hyperparathyroidism surgery. The method of the present invention can reduce the detection time to 20 minutes, greatly shortening the operation time and assisting in determining whether the hyperparathyroid glands have been completely removed.

[0004] The first object of the present invention is to provide a method for quickly predicting surgical results in hyperparathyroidism surgery, comprising the following steps:

[0005] Blood samples are collected before and after hyperparathyroidism removal surgery, and the parathyroid hormone content in the blood samples is detected using a detection product. The surgical effect prediction result (i.e., whether the hyperparathyroidism removal is complete) is obtained based on the changes in the parathyroid hormone content before and after the surgery;

[0006] The detection product contains anti-parathyroid hormone antibodies, which are secreted by a hybridoma cell line with a preservation number of CCTCC No. 2025122.

[0007] Furthermore, the detection product contains an antibody pair, which includes a labeled antibody and a detection antibody. The labeled antibody is an anti-parathyroid hormone antibody secreted by a hybridoma cell line with a preservation number of CCTCC No. 2025122, and the detection antibody is antibody FAB-M003-4H3.

[0008] Furthermore, the labeled antibody contains a covalently linked marker and antibody, and the detection antibody binds to the antigen in the sample to be tested through the labeled antibody, and utilizes the characteristics of the marker to detect the presence and content of the antigen.

[0009] Furthermore, post-operatively refers to 10 minutes or even longer after the operation, such as 10 minutes, 20 minutes, 30 minutes or more after the operation.

[0010] The second object of the present invention is to provide a hybridoma cell line, wherein the hybridoma cell line is hybridoma cell line PTH101, and the deposit number is CCTCC No. 2025122.

[0011] A third object of the present invention is to provide a monoclonal antibody secreted by the hybridoma cell line. The antibody comprises a variable region and a constant region, wherein the variable region comprises a heavy chain variable region and a light chain variable region, and the constant region comprises a heavy chain constant region and a light chain constant region, and the CDRs and FRs comprise complementarity determining regions.

[0012] The fourth object of the present invention is to provide a polynucleotide encoding the monoclonal antibody.

[0013] The fifth object of the present invention is to provide a recombinant expression vector containing the polynucleotide.

[0014] Furthermore, expression vectors include but are not limited to DNA, RNA, adeno-associated virus vectors, plasmids, and the like.

[0015] The sixth object of the present invention is to provide a recombinant cell containing the recombinant expression vector.

[0016] Furthermore, the host cell can be a prokaryotic cell or a eukaryotic cell, such as an animal cell (CHO cell, 293T cell, etc.), a microorganism (fungus, bacteria), etc.

[0017] The seventh object of the present invention is to provide a monovalent antibody, a bivalent antibody, a multivalent antibody or a recombinant protein containing the monoclonal antibody.

[0018] Furthermore, the recombinant protein contains

[0019] (a) the monovalent antibody, bivalent antibody or multivalent antibody;

[0020] (b) Tag sequences that facilitate expression and / or purification.

[0021] Preferably, the tag sequence includes an Fc tag, an HA tag, or a 6xHis tag. For example, if the fusion protein is formed with an Fc fragment, the structure of the fusion protein from the N-terminus to the C-terminus is as shown in Formula Ia or Ib:

[0022] ALB(Ia),

[0023] BLA(Ib),

[0024] Wherein A is the above-mentioned antibody, B is the Fc fragment of IgG, and L is no or a flexible linker. Preferably, the flexible linker is a peptide linker.

[0025] An eighth object of the present invention is to provide a detection product comprising the hybridoma cell line, monoclonal antibody, polynucleotide, recombinant expression vector, recombinant cell, monovalent antibody, bivalent antibody, multivalent antibody, or recombinant protein. The product may be in the form of, but not limited to, a reagent, a detection plate, or a kit.

[0026] Furthermore, the detection products include but are not limited to detection products using methods such as double antibody sandwich ELISA, blocking ELISA, indirect ELISA, competitive ELISA, time-resolved fluorescence, fluorescent immunochromatography or colloidal gold.

[0027] Furthermore, the detection product contains a labeled antibody and a detection antibody, the labeled antibody is an anti-parathyroid hormone antibody secreted by a hybridoma cell line with a preservation number of CCTCC No. 2025122, and the detection antibody is antibody FAB-M003-4H3.

[0028] Furthermore, the detection product includes a fluorescent immunochromatography product, which contains a sample pad, a conjugate pad, a detection line and a quality control line. The conjugate pad contains a labeled antibody (containing a fluorescent marker), the detection line is fixed with a detection antibody, and the quality control line is fixed with a secondary antibody.

[0029] A ninth object of the present invention is to provide a method for detecting PTH for non-diagnostic purposes (application of the detection product), comprising the following steps:

[0030] S1. Constructing an immunochromatographic detection system using the labeled antibody and the detection antibody; wherein the labeled antibody is modified with an identifiable marker (such as fluorescence);

[0031] S2. Using the detection system of step S1, detecting parathyroid hormone solutions of known different concentrations, and collecting identifiable labeled signals;

[0032] S3. Establishing a standard curve between the signal (such as intensity) of the identifiable marker and the parathyroid hormone content according to step S2;

[0033] S4. Take the sample to be tested, and perform detection using the detection system of step S1. Substitute the obtained identifiable labeled signal into the standard curve of step S3 to calculate the content of parathyroid hormone in the sample to be tested.

[0034] The above-described method can detect PTH in actual blood samples, achieving a sensitivity of 0.11 pg / mL. It should be noted that the present invention provides the detection sensitivity of actual samples, not the sensitivity under ideal conditions. It is well known in the art that many factors can affect the sensitivity of actual blood samples, such as sample storage, sample quality, and reagent quality. The fact that the present invention can achieve such excellent sensitivity in actual samples is sufficient to demonstrate its advantages.

[0035] The tenth object of the present invention is to provide the use of the hybridoma cell pair, monoclonal antibody pair, hybridoma cell line, monoclonal antibody, polynucleotide, recombinant expression vector, recombinant cell, monovalent antibody, bivalent antibody, multivalent antibody, recombinant protein or detection product in the preparation of parathyroid hormone detection products.

[0036] Beneficial effects of the present invention:

[0037] (1) The present invention is based on the fact that there is no means on the market that can quickly assist in determining whether hyperparathyroidism has been completely removed in the operating room. A new method for quickly predicting the surgical outcome during hyperparathyroidism surgery has been developed. Specifically, the existing test for serum parathyroid hormone requires drawing blood and sending it to the laboratory, where the serum is tested after centrifugation, which takes 45 minutes to 1 hour. However, the use of the self-made test product of the present invention can quickly and accurately detect changes in PTH content in blood samples. The detection time using the existing method is reduced to 20 minutes, greatly shortening the operation time.

[0038] (2) The test product of the present invention contains anti-PTH antibodies secreted by specific hybridoma cells. The inventors screened for high-titer anti-PTH antibodies using PTH-immunized mice. The screened antibodies had titers greater than 10 million. They then paired these high-titer antibodies to find suitable antibody pairs for detection. Ultimately, they found that the combination of antibody 101 and H43, particularly antibody 101 as the labeling antibody and antibody H43 as the detection antibody, achieved the best results, with a sensitivity of 0.11 pg / ml (actual serum sample).

[0039] Biomaterial Deposit

[0040] Hybridoma cell line PTH101 (Hybridoma cell line PTH101), the hybridoma cell line PTH101 was deposited in the China Center for Type Culture Collection on April 8, 2025, with the deposit number CCTCC No.2025122, and the deposit address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of a cross-section of the fluorescent immunochromatographic test paper along the length direction in Example 4;

[0042] Figure 2 Schematic diagram of the fluorescent immunochromatographic test paper in Example 4. The reference numerals in the figure are: 1-base plate, 2-nitrocellulose membrane, 3-absorbent paper, 4-sample pad, 5-conjugate pad, 6-quality control strip, 7-detection strip.

[0043] Figure 3 This is the standard curve for detecting PTH by fluorescence immunochromatography in Example 4.

[0044] Figure 4 This is the correlation curve for quantitative detection of PTH concentration in human blood by fluorescent immunochromatography and CLIA method in Example 4.

[0045] Figure 5 A portable device used for POCT testing. DETAILED DESCRIPTION

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0047] Explanation of terms involved in the following embodiments:

[0048] In the present invention, the terms "antibody of the present invention", "monoclonal antibody of the present invention", "anti-PTH antibody of the present invention", etc. have the same meaning and are used interchangeably, and all refer to antibodies that specifically recognize and bind to PTH.

[0049] For purposes of the present invention, the term "antibody" refers to a protein composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, with the number of disulfide bonds varying between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is aligned with the first constant region of the heavy chain, and the variable region of the light chain is aligned with the variable region of the heavy chain. Of course, the present invention is protected in the form of a hybridoma cell line. If necessary, a person skilled in the art can sequence the cell line, and the sequence remains within the scope of protection of the present invention.

[0050] In the present invention, "multivalent" refers to a fusion protein comprising multiple variable regions of the antibodies of the present invention or the antibodies of the present invention.

[0051] In the present invention, the term "variable" means that certain parts of the variable region in an antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the entire antibody variable region. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a beta-fold configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial beta-fold structure. The CDRs in each chain are closely together through the FR region and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0052] In the present invention, the terms "heavy chain variable region" and "VH" are used interchangeably, and the terms "light chain variable region" and "VL" are used interchangeably.

[0053] In the present invention, the terms "antibody of the present invention," "protein of the present invention," or "polypeptide of the present invention" are used interchangeably to refer to polypeptides that specifically bind to PTH, such as proteins or polypeptides having a heavy chain variable region and a light chain variable region. These may or may not contain an initial methionine.

[0054] Generally, an antibody's antigen-binding properties are described by three specific regions within the variable region of the heavy chain, known as the variable regions (CDRs). This region is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a loop structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0055] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0056] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention can be: (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; (ii) a polypeptide having a substituent group in one or more amino acid residues; (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol) or a polypeptide (e.g., a polypeptide that extends the half-life of the polypeptide, such as an engineered antibody Fc domain); or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, a sequence for purifying the polypeptide, a proprotein sequence, or a fusion protein with a 6xHis tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0057] The antibodies of the present invention refer to polypeptides having PTH binding activity and comprising the aforementioned CDR regions or variable regions. The term also encompasses variants of polypeptides comprising the aforementioned CDR regions or variable regions that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids, as well as the addition of one or more amino acids (generally within 20, preferably within 10, and more preferably within 5) to the C-terminus and / or N-terminus. For example, substitutions with amino acids having similar or similar properties generally do not alter protein function. For another example, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter protein function. The term also encompasses active fragments and active derivatives of the antibodies of the present invention.

[0058] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0059] The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof.

[0060] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention.

[0061] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0062] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0063] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0064] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0065] Once the relevant sequence is obtained, recombinant methods can be used to obtain it in large quantities. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.

[0066] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into protein sequences of the present invention by chemical synthesis.

[0067] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0068] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include Escherichia coli, Streptomyces, bacterial cells of Salmonella typhimurium, fungal cells such as yeast, and animal cells such as CHO, COS7, and 293 cells.

[0069] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated using the CaCl2 method, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0070] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0071] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0072] Labeled antibodies

[0073] The antibodies of the present invention may be used alone or, in conjunction with or coupled to a detectable marker (for diagnostic purposes), in a related assay.

[0074] Detectable labels for diagnostic purposes include, but are not limited to, colloidal gold labels, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0075] Detection method

[0076] The present invention also relates to a method for detecting PTH. The method generally comprises the following steps: obtaining a sample to be tested; dissolving the sample in a medium; and detecting the level of a labeled signal in the dissolved sample to infer the level of PTH.

[0077] In the detection method of the present invention, the sample used is not particularly limited.

[0078] Reagent test kit

[0079] The present invention also provides a kit containing the antibody (or fragment thereof) or detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc.

[0080] Preferably, the present invention also provides a detection kit for detecting PTH levels, which includes a capture antibody and a detection antibody, a medium for dissolving the sample, and general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc.

[0081] Example 1 Preparation of anti-PTH monoclonal antibody hybridoma cell line

[0082] 1. PTH immunogen

[0083] The invention uses the human parathyroid hormone (PTH) recombinant protein made in the laboratory as the immunogen, which is an Escherichia coli expression product.

[0084] 2. Immunization of Mice

[0085] Five 8-week-old female BALB / c mice were immunized. Newly purchased BALB / c mice were housed in an animal barrier environment for one week to allow them to acclimate. Blood was collected from the tail tip of the mice using a capillary tube into a 0.6 ml centrifuge tube, incubated overnight at 4°C, and centrifuged at 2,000 rpm for 10 minutes. The supernatant was collected and used as the negative control serum. For the first immunization, PTH antigen was mixed with equal volumes of Freund's complete adjuvant and thoroughly emulsified using a dual-channel syringe emulsifier to form an oil-in-water emulsion. The emulsion was injected subcutaneously at multiple points on the back of the neck to immunize the mice. The immunization dose was 100 μg / mouse. Two weeks later, a second immunization was performed at a dose of 100 μg / mouse. PTH antigen was mixed with an equal volume of Freund's incomplete adjuvant and emulsified. The emulsion was injected subcutaneously at multiple points on the back of the neck or intraperitoneally. Two weeks later, a third immunization was performed using the same procedures as the second immunization. One week later, blood was collected from the tail tip, and the antibody titer of the sera from the immunized mice was determined by ELISA. Immunized mice with the highest antibody titers were selected and, one week later, received a pulse immunization with an intraperitoneal injection of 200 μg of antigen without Freund's adjuvant. Three days after the pulse immunization, spleens were harvested for cell fusion experiments.

[0086] 3. Cell Fusion

[0087] One day before the cell fusion experiment, two ICR mice were sacrificed by cervical dislocation. Peritoneal macrophages were extracted using HAT-containing medium, with 100 μl / well plated onto six 96-well plates. Three days after the shock immunization, the mice were sacrificed by cervical dislocation. The spleens were removed and gently triturated using a 10 ml syringe cartridge. The cells were filtered through a 70 μm cell strainer and centrifuged at 1,000 rpm for 8 minutes. The spleen cells were washed once with 20 ml of serum-free medium and counted. Sp2 / 0 mouse myeloma cells in logarithmic growth phase were collected from ten 10 cm culture dishes and counted. The two cell suspensions were mixed at a ratio of 5:10 spleen cells to Sp2 / 0 cells, and the cells were centrifuged at 1,200 rpm for 6 minutes. The supernatant was discarded, and the bottom of the centrifuge tube was gently tapped to evenly disperse the cell pellet. Add 1 ml of PEG1450 (Sigma-Aldrich, P7181) to initiate cell fusion. Terminate fusion by adding 15 ml of serum-free medium. Incubate at 37°C for 10 minutes and centrifuge at 800 rpm for 6 minutes. Discard the supernatant and resuspend the cells in DMEM supplemented with HAT and 20% imported serum. Add 100 μl / well to a 96-well plate containing feeder cells and culture at 37°C in a 5% CO2 incubator.

[0088] 4. Screening and Subcloning of Positive Cells

[0089] Observe the cells on the 5th day after fusion and replace the medium with DMEM medium containing HT and 10% imported serum (200μl / well). On the 7th day, ELISA was used to detect the antibody content in the cell supernatant and screen the positive hybridoma cells. Subclone the positive cells by limiting dilution, resuspend the cell pellet by pipetting, add it to a 96-well plate containing feeder cells according to a 5-fold gradient dilution, and detect the antibody content in the supernatant after 6 days. Select monoclones and repeat 3 to 5 rounds of monoclonal cloning. The selected monoclonal hybridoma cells were expanded to 12-well, 6-well, 6 cm culture dishes, and 10 cm culture dishes, respectively, and frozen in liquid nitrogen.

[0090] 5. ELISA to detect PTH antibody titer

[0091] (1) PTH recombinant protein (1 mg / ml) was diluted to 1 μg / ml with 0.05 M carbonate buffer (pH 9.6), 100 μl / well, and incubated at 4°C overnight. (2) Abandon the antigen, add 200 μl of washing solution to each well, shake and pat dry, and repeat twice. Add blocking solution containing 2% BSA, 150 μl / well, and incubate at 37°C for 2 hours. (3) Add 100 μl of hybridoma cell supernatant containing PTH antibody to each well, and incubate at 37°C for 2 hours. (4) Wash 3 times, add diluted Eu 3+ Labeled goat anti-mouse secondary antibody, 100 μl / well, incubate at 37℃ for 1 hour. (5) Wash 5 times, add 100 μl enhancement solution to each well, shake at room temperature for 5 minutes, and detect fluorescence values.

[0092] Example 2 Preparation of PTH Monoclonal Antibody

[0093] 1. Ascites Preparation

[0094] Ten-week-old female BALB / c mice were pre-stimulated by intraperitoneal injection of 0.5 ml of pristane (Sigma-Aldrich, P9622). Seven days later, 2-3 × 10 6 Hybridoma cells were cultured. After 7 to 10 days, the mouse's abdomen became noticeably distended and its movements became sluggish. The mouse's peritoneal cavity was punctured with a 20 ml syringe needle, and the ascites was drained by gravity. The ascites was harvested and centrifuged at 4,000 rpm for 10 minutes. The supernatant was frozen at -20°C. After an interval of 2 days, ascites was collected again. ELISA assay determined that the titer of the ascites antibody was greater than 10 million.

[0095] 2. Ascites Antibody Purification

[0096] Ascites antibodies were purified by protein G affinity chromatography. The specific steps were as follows: ascites fluid frozen at -20°C was thawed in a water bath, diluted with 8 volumes of PBS, and filtered through a 0.45 μm filter. A gravity column was prepared using 5 ml of protein G affinity filler (Hangzhou Newlong Biotechnology). After equilibration with PBS, the diluted ascites fluid was loaded onto the column. The column was rinsed with PBS until equilibrium was reached, and then eluted with 0.1 M glycine-HCl (pH 2.7). The eluate was collected with 10× neutralization buffer (1 M Tris-HCl, pH 9.0). The protein concentration of each tube was determined, and the 2 to 3 tubes with the highest concentrations were combined and dialyzed overnight. The antibody titer was determined to be greater than 10 million.

[0097] Example 3 Antibody Pairing Evaluation

[0098] The two PTH antibodies 101 and 234 with the highest titers were taken and compared with time-resolved fluorescent microspheres (containing Eu 3+The commercial PTH antibodies (3AG358, 4H3, 5G4, and Medix) were conjugated to nitrocellulose membranes using ACME microspheres (labeled with time-resolved fluorescence microspheres, EU300-10) for pairing. The antibodies were then sprayed onto nitrocellulose membranes and assembled into fluorescent immunochromatographic test strips for pairing screening. The results are shown in Table 1. The commercial PTH antibodies 3AG358 and 4H3 showed good pairing with the homemade antibody 101. The 4H3 antibody exhibited lower background and a wider linear range. Therefore, the PTH antibody FAB-M003-4H3 (https: / / shops.fantibody.com / products / pth-antibody?_pos=1&_sid=f90907052&_ss=r) from Fantibody was paired with the homemade antibody 101 for fluorescent immunochromatographic test strip preparation. The hybridoma cell line secreting antibody 101 was also deposited.

[0099] Table 1 PTH antibody pairing screening results

[0100]

[0101]

[0102] Note: The unit of PTH standard (10,000 and 30,000): pg / ml

[0103] Example 4 Preparation of Fluorescent Immunochromatographic Test Paper for Quantitative Detection of PTH in Human Blood 1. Research Subjects:

[0104] The specimens were collected from 15 patients undergoing parathyroidectomy surgery at Jiangyuan Hospital. Blood was drawn with a syringe and the samples were kept for future use.

[0105] 2. Reagents and instruments:

[0106] The immunochromatographic test strips were divided into experimental and control groups. The labeled antibody in the experimental group was 101 monoclonal antibody, and the detection antibody was 4H3 monoclonal antibody. The control group used a Roche test kit. The quality control antibody (sheep anti-chicken IgY), fluorescent microspheres, and fluorescence detector for the immunochromatographic test strips were from Wuxi Jiangyuan Industrial Technology and Trade Co., Ltd. The nitrocellulose membrane was from Merck-Millipore, USA. The sample pad, conjugate pad, base plate, and absorbent paper were purchased from Shanghai Jieyi Company. The CLIA test kit was a Roche product. All other reagents were domestically produced analytical grade.

[0107] 3 Preparation method:

[0108] 3.1 Antibody-labeled fluorescent microspheres:

[0109] The fluorescent microspheres were washed with MES activation buffer at pH 7.2-7.6, and carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added and reacted at room temperature for a certain time. The fluorescent microspheres were washed and re-dissolved with 0.05M phosphate buffer at pH 7.2-7.6, and the labeled antibody was added and reacted at room temperature for 2 hours. 0.05M phosphate buffer at pH 7.2-7.6 containing 10% BSA was added and blocked at room temperature for 30 minutes. The fluorescent microspheres were washed and re-dissolved to the original volume with 1% BSA, 0.1% Tween-20, and 0.05M phosphate buffer at pH 7.2-7.6. The microspheres were quantitatively sprayed onto the conjugate pad, dried at 35-38°C in the dark for 1 hour, and sealed with a desiccant for later use.

[0110] 3.2 Fluorescent immunochromatographic test strip assembly component processing:

[0111] (1) Sample pad processing

[0112] The samples were immersed in 0.02 M pH 7.4 phosphate buffer containing 1% BSA and 0.1% Triton 100 and dried.

[0113] (2) Preparation of nitrocellulose membrane

[0114] Use 0.02M pH 7.4 phosphate buffer containing 1% sucrose to dilute the detection antibody and quality control antibody to 1 mg / mL respectively. Spray them onto nitrocellulose membrane with a 0.5 cm interval. After drying, add desiccant and seal for later use;

[0115] 3.3 Assembly of fluorescent immunochromatographic test strips

[0116] In an environment with a humidity of less than 35% and a stable temperature of 20-25°C, a nitrocellulose membrane 2, a conjugate pad 5 labeled with fluorescent microspheres, a sample pad 4, and absorbent paper 3 are attached to a PVC base plate 1 to form a microfiltration system. The system is then cut into 0.4 cm wide pieces and placed in a cartridge to form a test strip ( Figure 1 and Figure 2 ).

[0117] 4. Detection methods:

[0118] (1) Sampling: Use a syringe to draw 4 mL of blood sample into a heparin tube.

[0119] (2) Sample pretreatment: The blood collection tube from which the blood sample was drawn was centrifuged at 4,000 rpm for 3 min before use.

[0120] (3) Sample addition: Take 30 μL of the centrifuged blood sample to be tested and add it to the sample well of the fluorescent immunochromatographic test paper, then add 60 μL of sample diluent, and react in an incubator at 37°C for 15 minutes.

[0121] (4) Detection: Import the standard curve into the portable fluorescent immunoassay quantitative analyzer in advance, insert the reacted fluorescent immunochromatographic test strip into the card slot of the quantitative analyzer, run the instrument, and the instrument automatically reads the card to give the PTH value in the fluorescent immunochromatographic test strip.

[0122] (5) Preparation of standard curve: Prepare PTH standard at 6 different concentrations, namely 0 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, 500 pg / mL, and 1000 pg / mL, and make 3 parallel samples for each concentration.

[0123] (6) Fluorescent immunochromatographic test strip performance test: (a) Sensitivity: Measure 10 blank samples, take the average value (x) and standard deviation (s), calculate x ± s, and use this value to find the corresponding dose on the standard curve. (b) After the test strips are stored at 4°C in the dark for 6 months, PTH fluorescent immunochromatographic test strips from the same batch and different batches are drawn and tested with a standard at a concentration of 100 pg / mL. The intra-assay and inter-assay CVs are calculated. (c) The standard is prepared at 6 concentrations corresponding to the PTH standard curve for specificity detection.

[0124] (7) Comparison with the electrochemiluminescence (CLIA) test kit: Strictly following the instructions of the CLIA test kit, blood samples from 15 patients were tested in parallel with the fluorescent test strips.

[0125] (8) Statistical analysis: SPSS 19.0 statistical software was used to analyze the data. The chi-square test was used between groups, and a P value less than 0.05 was considered statistically significant. Paired T-tests were used to compare correlations and differences.

[0126] 5. Results and Analysis:

[0127] (1) Interpretation of test results: During the test, the liquid moves forward due to the chromatographic effect. If the PTH content in the sample is too low, the PTH in the sample will combine with the fluorescent microspheres bound to the labeled antibody on the conjugate pad to form a relatively small amount of complex C1. The labeled antibody in the conjugate pad will bind to the quality control antibody on the C line in large quantities. Therefore, the fluorescence of the T line will be much lighter than that of the C line or no fluorescence will be detected at all, indicating that the PTH content is very low. If the concentration of PTH in the sample is high, the complex C1 will be relatively large, binding to the detection antibody at the T line and forming a large amount of antibody-antigen-antibody complex C2. Moreover, the higher the PTH in the sample, the darker the fluorescence value of the T line, and the higher the PTH content. If there is no fluorescence value on the C line, the PTH result is invalid and the test strip needs to be replaced for retesting.

[0128] (2) Standard curve drawing: According to the statistical method, the fluorescence value signal of the detected sample is used as the vertical axis and the concentration of the PTH standard is used as the horizontal axis (Table 2 is the data of the experimental group), and an equation is established and fitted into a standard curve. The R 2 The R value of the standard curve of the control group was 0.9985, which was a good linearity and met the requirements of quantitative detection. 2 The linearity is 0.9880, which is poor compared with the experimental group.

[0129] Table 2 PTH standard test results

[0130]

[0131]

[0132] (3) Performance evaluation of PTH immunochromatographic fluorescence test strips: (a) Sensitivity: The mean reading at the zero dose point of the test group was 0.04, which was converted to 0.11 pg / mL on the standard curve. Samples of 0.20 pg / mL, 1 pg / ml, 2 pg / mL, 4 pg / mL, and 8 pg / mL were taken for testing, and the concentration was converted using the standard curve. It was found that this series of PTH samples with a concentration gradient could all be accurately detected. The control group was verified using the same method, and the sensitivity could only reach 2.0 pg / mL, which was an order of magnitude different from the control group. (b) Stability and precision: The CV values ​​corresponding to each group of the standard curve of the test group were all less than 15% (Table 2). When the test strips were stored at 4°C in the dark for 6 months, the intra-assay and inter-assay CVs were less than 15%, indicating that the test strips had good stability and precision. The control group was not much different from the test group in terms of stability and precision. (c) Specificity: The prepared PTH standard and thyroxine standard were tested simultaneously using the test strips in the experimental group. The cross-reaction rate (CR%) at each concentration point was calculated as follows: measured thyroxine value / measured PTH value × 100%. Within the above concentration range, the cross-reaction rate with thyroxine was less than 0.1%, indicating no cross-reaction and good specificity. When the control group test strips were used, the cross-reaction rate with thyroxine exceeded 3% at low concentrations, indicating significantly lower specificity than the test group.

[0133] (4) Comparison with CLIA method: Plasma and serum samples from 15 patients were tested using CLIA kits, experimental group fluorescent immunochromatographic test strips, and control group immunochromatographic test strips, respectively. The upper limit of the PTH value of 65 pg / mL was taken as the cutoff value of this kit, and the data were analyzed. The results showed that the correlation coefficient between the experimental group immunochromatographic test strips and the CLIA kit was 0.9996, and the correlation curve was Y = 1.0001X + 0.5969, where Y is the PTH concentration (pg / mL) obtained by the fluorescent immunochromatographic test strips of the present invention, and X is the PTH concentration (pg / mL) obtained by the CLIA kit. It can be seen that the correlation between the two is very good. The correlation coefficient between the control group immunochromatographic test strips and the CLIA kit was 0.9888, which is poorer than that of the experimental group.

[0134] Table 3 Comparison of the results of quantitative detection of PTH content in human blood by fluorescence immunochromatography

[0135]

[0136]

[0137] The above-mentioned point-of-care (POCT) tests are based on venous blood drawn 20 minutes after surgery. In practical applications, venous or arterial blood can be drawn before skin incision and at 0, 10, or 20 minutes after removal of the affected parathyroid gland during hyperparathyroidism surgery. The same blood type must be used. For primary hyperparathyroidism, if the parathyroid hormone level 10 minutes after removal of the hyperfunctioning parathyroid gland is 50% or more lower than the highest value before skin incision or gland removal, it is considered that all hyperfunctioning parathyroid glands have been removed. For hyperparathyroidism after renal failure, if the parathyroid hormone level 10 minutes after the removal of the hyperfunctioning parathyroid glands decreases by more than 80% compared with the highest value before skin incision or gland removal, it is considered that all hyperfunctioning parathyroid glands have been removed (Thyroid and Metabolic Surgery Group of the Chinese Medical Association's Surgery Branch, Parathyroid and Bone Metabolic Disease Professional Committee of the Chinese Association of Research Hospitals. Chinese Expert Consensus on the Perioperative Management of Primary Hyperparathyroidism (2020 Edition) [J]. Chinese Journal of Practical Surgery, 2020, 40(6): 634-638.). Intraoperative detection is by fluorescent immunochromatographic test strips for quantitative detection of human parathyroid hormone in plasma or serum, and by portable instruments ( Figure 5 ), completed in the operating room.

[0138] From the above examples and investigation results, it can be seen that the monoclonal antibodies prepared using the two PTH epitope peptides of the present invention can specifically recognize PTH. The fluorescent immunochromatographic POCT quantitative detection method prepared using the same can accurately quantify PTH in a short time, and the results are not significantly different from those obtained by CLIA detection. It fully meets the requirements of clinical application, is simple to operate, small in size, easy to carry, and easy to store, and is worthy of clinical promotion and application.

[0139] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for rapidly predicting surgical results in hyperparathyroidism surgery, characterized in that: The following steps are involved: Blood samples are collected before and after hyperparathyroidism removal surgery, and the parathyroid hormone content in the blood samples is detected using a detection product. The surgical effect prediction result is obtained based on the changes in the parathyroid hormone content before and after the surgery; The detection product contains anti-parathyroid hormone antibodies, which are secreted by a hybridoma cell line with a preservation number of CCTCC No. 2025122.

2. The method according to claim 1, characterized in that The detection product contains a labeled antibody and a detection antibody, wherein the labeled antibody is an anti-parathyroid hormone antibody secreted by a hybridoma cell line with a preservation number of CCTCC No. 2025122, and the detection antibody is antibody FAB-M003-4H3.

3. A hybridoma cell line, characterized in that The hybridoma cell line is the hybridoma cell line PTH101, and its preservation number is CCTCC No. 2025122.

4. A monoclonal antibody, characterized in that The monoclonal antibody is secreted by the hybridoma cell line according to claim 3. A polynucleotide encoding the monoclonal antibody according to claim 4 .

6. A recombinant expression vector carrying the polynucleotide according to claim 5.

7. A recombinant cell containing the recombinant expression vector according to claim 6.

8. A monovalent antibody, a bivalent antibody, a multivalent antibody or a recombinant protein comprising the monoclonal antibody according to claim 4.

9. A detection product, characterized in that: The detection product contains the hybridoma cell line according to claim 3, the monoclonal antibody according to claim 4, the polynucleotide according to claim 5, the recombinant expression vector according to claim 6, the recombinant cell according to claim 7, or the monovalent antibody, bivalent antibody, multivalent antibody or recombinant protein according to claim 8.

10. The detection product according to claim 9, characterized in that: Contains at least one of the following characteristics: (1) The detection products include detection products using double antibody sandwich ELISA, blocking ELISA, indirect ELISA, competitive ELISA, time-resolved fluorescence, fluorescence immunochromatography or colloidal gold methods; (2) The detection product contains a labeled antibody and a detection antibody, wherein the labeled antibody is an anti-parathyroid hormone antibody secreted by a hybridoma cell line with a deposit number of CCTCC No. 2025122, and the detection antibody is antibody FAB-M003-4H3; (3) The detection product contains a sample pad, a conjugate pad, a detection line and a quality control line. The conjugate pad contains a labeled antibody, the detection antibody is fixed on the detection line, and the secondary antibody is fixed on the quality control line.

11. Medical use of the hybridoma cell line according to claim 3, the monoclonal antibody according to claim 4, the polynucleotide according to claim 5, the recombinant expression vector according to claim 6, the recombinant cell according to claim 7, the monovalent antibody, bivalent antibody, multivalent antibody or recombinant protein according to claim 8, or the detection product according to claim 9 or 10, characterized in that: The medical use includes at least one of the following: (1) For the preparation of parathyroid hormone detection products; (2) Used to prepare hyperparathyroidism detection products.

12. A method for detecting parathyroid hormone for non-diagnostic purposes, characterized in that: The following steps are involved: S1. Construct an immunochromatographic detection system using a labeled antibody and a detection antibody; wherein the labeled antibody is an anti-parathyroid hormone antibody secreted by a hybridoma cell line with a deposit number of CCTCC No. 2025122, and the detection antibody is antibody FAB-M003-4H3, and the labeled antibody is modified with a recognizable label; S2. Using the detection system of step S1, detecting parathyroid hormone solutions of known different concentrations, and collecting identifiable labeled signals; S3, establishing a standard curve of the identifiable marker signal and the parathyroid hormone content according to step S2; S4. Take the sample to be tested, and perform detection using the detection system of step S1. Substitute the obtained identifiable labeled signal into the standard curve of step S3 to calculate the content of parathyroid hormone in the sample to be tested.

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