Humanized monoclonal antibody for detecting human-synuclein oligomer and application thereof

Abstract

The invention provides a humanized monoclonal antibody for detecting a human-synuclein oligomer and application of the humanized monoclonal antibody, and belongs to the technical field of biological engineering. The light chain CDR sequence of the binding part comprises at least one of LCDR1, LCDR2 and LCDR3, and the amino acid sequence of the LCDR1 is as shown in SEQ ID NO: 1; the amino acid sequence of the LCDR2 is as shown in SEQ ID NO: 2, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO: 3. The monoclonal antibody is used as a raw material, and a unique repeated epitope double-antibody sandwich immunodetection method is adopted, so that the human a-Syn oligomer prepared in vitro or in a sample can be detected with high specificity, high sensitivity and high accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to a humanized monoclonal antibody for detecting human-synuclein oligomers and an application thereof. Background Art

[0002] α-Synuclein (α-Syn) is a 140-amino acid protein that normally exists primarily as a soluble monomer in presynaptic terminals of neurons. However, under certain pathological conditions, α-Syn misfolds and aggregates into fibrillar aggregates, which deposit in neurons or oligodendrocytes, forming characteristic pathological structures such as Lewy bodies (LBs) in neurons and glial cytoplasmic inclusions (GCIs). LBs are primarily found in the brains of patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB), while GCIs are primarily found in the brains of patients with multiple system atrophy (MSA). They serve as important pathological markers for the diagnosis of these diseases.

[0003] Although a-Syn deposited in LB and GCI primarily exists as fibrils, oligomeric intermediates formed during its aggregation process are toxic to neurons. a-Syn oligomers not only directly damage neurons but also indirectly damage them by activating immune inflammatory responses. Furthermore, a-Syn oligomers can propagate within neural networks using a prion-like mechanism, leading to the continuous spread of a-Syn oligomer pathology in the patient's brain. Therefore, detecting a-Syn oligomers in tissues and body fluids is considered an important biomarker reflecting the neuropathology of PD, DLB, and MSA.

[0004] Currently, an important method for detecting α-Syn oligomers is a double-antibody sandwich immunoassay, using monoclonal antibodies that recognize the same epitope as the capture and detection antibodies, respectively. In this case, once an α-Syn monomer is bound by the capture antibody, it can no longer bind to the detection antibody that recognizes the same epitope, making it undetectable. However, after the α-Syn oligomer is bound by the capture antibody, there are still vacant epitopes available for the detection antibody to bind, allowing it to be detected and quantified. Currently, this method has been used to detect α-Syn oligomers in the cerebrospinal fluid, saliva, plasma, and red blood cells of patients with PD, DLB, and MSA, but its sensitivity is relatively low. The development of more sensitive methods for detecting α-Syn oligomers is of great significance for the diagnosis of α-Syn pathology. Humanization of monoclonal antibodies will significantly improve the sensitivity and specificity of detecting protein markers in patient samples. Based on the preparation of a murine α-Syn monoclonal antibody, the antibody is humanized and then detected and validated using a double-antibody sandwich method in vitro and in real samples. Summary of the Invention

[0005] The purpose of the present invention is to provide a humanized monoclonal antibody for detecting human α-synuclein oligomers and its application. Using this monoclonal antibody as the raw material and a unique repeat epitope double antibody sandwich immunoassay, human α-Syn oligomers prepared in vitro or in samples can be detected with high specificity, high sensitivity and high accuracy.

[0006] The technical solution of the present invention is achieved as follows: The present invention provides a humanized monoclonal antibody for detecting human α-synuclein oligomers, wherein the light chain CDR sequence of the binding portion includes at least one of LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 1; the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 3.

[0007] As a further improvement of the present invention, the heavy chain CDR sequence of the binding portion includes at least one of HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO:4; the amino acid sequence of the HCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:6.

[0008] As a further improvement of the present invention, the preparation method of the human α-synuclein oligomer is as follows: diluting the human α-synuclein polypeptide with a PBS buffer solution, incubating at a constant temperature with shaking, then adding sodium dodecyl sulfate, sonicating, centrifuging, collecting the supernatant, and freeze-drying to obtain human α-synuclein oligomers.

[0009] As a further improvement of the present invention, the constant temperature oscillation incubation temperature is 36-38° C., the oscillation frequency is 500-1000 rpm, and the time is 60-80 h.

[0010] As a further improvement of the present invention, the ultrasonic time is 20-40 minutes, and the centrifugal temperature is 2-5° C. and the time is 20-40 minutes.

[0011] As a further improvement of the present invention, the pH value of the PBS buffer is 7-7.7, the dilution concentration is 3-7 mg / mL, and the final concentration of the sodium lauryl sulfate is 2-4 wt %.

[0012] The present invention further protects a method for specifically detecting α-synuclein oligomers using the above-mentioned humanized monoclonal antibody for detecting human α-synuclein oligomers, comprising: combining the antibody with a solid phase carrier and a label, respectively, to prepare a solid phase antibody and a labeled antibody, adding the sample for incubation, and generating a signal.

[0013] The present invention further protects a detection kit prepared using the above-mentioned humanized monoclonal antibody for detecting human α-synuclein oligomers as a raw material.

[0014] The present invention further protects the use of the above-mentioned detection kit in auxiliary diagnosis of Parkinson's disease.

[0015] The present invention has the following beneficial effects: The present invention provides a method for preparing and applying a humanized monoclonal antibody that specifically recognizes α-Syn. Using this monoclonal antibody as a raw material, a unique double-antibody sandwich immunoassay with repeated epitopes can detect human α-Syn oligomers prepared in vitro or in samples with high specificity, sensitivity, and accuracy.

[0016] The present invention also provides a kit for detecting human a-Syn oligomers, which can quantitatively detect a-Syn oligomers in a variety of patient samples (not limited to cerebrospinal fluid, blood, saliva, exosomes, etc.), and can be used for auxiliary diagnosis of PD, DLB, and MSA. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is the electrophoresis diagram of monoclonal antibody 4C3; Figure 2 is a titer chart of anti-α-synuclein oligomer monoclonal antibodies; Figure 3 This is the Western result of monoclonal antibody 4C3; Figure 4 This is a comparison chart of antibody activity; Figure 5 Figure 1 is the result of the test specificity; Figure 6 Comparison chart between normal people and PD patients Figure 7 This is a correlation analysis chart of plasma and saliva α-synuclein oligomers. DETAILED DESCRIPTION

[0019] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0020] Example 1, Preparation of α-synuclein oligomers:

[0021] 1. 5 mg of α-synuclein (prepared by KMD Biotechnology) was diluted to 5 mg / ml with PBS and then placed on a 37°C constant temperature shaker with an oscillation frequency of 800 rpm.

[0022] 2. After shaking for 72 h, add sodium lauryl sulfate to a final concentration of 3 wt% and sonicate for 30 min.

[0023] Centrifuge at 3.4°C, 10,000 rpm for 30 min. The supernatant is α-synuclein oligomers.

[0024] Example 2: Preparation of mouse monoclonal antibodies

[0025] 1. Animal immunization and polyclonal antibody titer determination: (1) Animal selection: Five healthy female Balb / c mice aged 5 weeks were selected and immunized with the immunogen prepared in Example 1.

[0026] (2) Immunization method: Immunization is performed by subcutaneous multiple-point injection. Freund's complete adjuvant is added for the first immunization, and incomplete adjuvant is used for the second immunization. The second immunization is performed three weeks after the first immunization, and then immunization is performed every two weeks, for a total of four immunizations.

[0027] (3) Immunization dose: 0.1 mg / animal / time.

[0028] (4) Collection of polyclonal antibody serum: Two weeks after the last immunization, blood was collected by tail cutting and diluted 100-fold with PBS. The serum was centrifuged at 3000 rpm for 10 min, and the supernatant was collected and aliquoted. The serum was stored at -20°C.

[0029] (5) The α-synuclein oligomers were coated on an enzyme-linked immunosorbent assay plate and then blocked. After adding polyclonal antibody serum and enzyme-labeled secondary antibody, a color reaction was performed to determine the titer of the polyclonal antibody. The results are shown in Table 1.

[0030]

[0031] According to the preliminary ELISA test results, the immune effect of mice numbered 2 was the best. According to the preliminary ELISA test results, the immune effect of mice numbered 2 was the best.

[0032] 2. Cell fusion and hybridoma cell screening:

[0033] (1) Recovery and culture of SP2 / 0 tumor cells. SP2 / 0 tumor cells were removed from the liquid nitrogen tank, dissolved in a 37°C water bath, transferred to a centrifuge bottle, and added with 37°C preheated RPMI1640 / 10. Centrifuged at 1000 rpm for 10 min, discarded the supernatant, and the cell clumps were broken up. 10 mL of RPMI1640 / 10 was added to the centrifuge bottle and mixed evenly. The cells were then placed in a 5% CO2, 37°C incubator for culture.

[0034] (2) Preparation of feeder cells. Take an unimmunized Bal b / c mouse, remove its eyeballs and collect blood. Then, dislocate the mouse by pulling its neck and immerse it in 75% alcohol. Carefully cut the mouse peritoneum with sterile forceps and scissors to fully expose the abdominal cavity. Use a 10mL sterile syringe to draw 10mL of cold HAT medium into the mouse's peritoneal cavity. Gently squeeze the mouse's peritoneal cavity with an alcohol cotton ball to withdraw the culture medium containing the feeder cells.

[0035] (3) Preparation of spleen cells. Take the first immunized Balb / c mouse with the best serum titer, remove the eyeball to collect positive blood, and kill the mouse by pulling the neck. Then, soak it in 75% alcohol. Then, remove the spleen of the mouse with the abdomen facing up. Grind the spleen with sterile scissors and forceps. Then, take 5 mL of preheated GNK wash solution and slowly add it to the nylon mesh to rinse the spleen cells to prepare a single cell suspension. Then transfer it to a 50 mL centrifuge tube and centrifuge at 1000 rpm for 10 minutes. Discard the supernatant, break up the cell clumps, and resuspend the cells for later use.

[0036] (4) Cell fusion and screening of positive hybridoma cells. SP2 / 0 tumor cells in the logarithmic growth phase were selected and transferred to a 50 mL centrifuge bottle. The supernatant was discarded after centrifugation to break up the cell clumps. Preheated GNK wash solution was added. The prepared spleen cells and tumor cells were mixed in the same centrifuge bottle at a ratio of 6:1-10:1. The cells were centrifuged and the supernatant was discarded to break up the cell clumps. The tube was placed in a 37°C water bath and 1 mL of preheated polymerizer PEG-1500 was added. The tube was shaken while adding. The tube was allowed to stand at 37°C for 5 min. GNK solution was added to 40 mL. The tube was centrifuged at 1200 rpm for 15 min. The supernatant was discarded to break up the cell clumps. 40 mL of preheated HAT selective medium was added to mix the cells evenly. The cells were added to the culture plate at 100 µL / well and cultured in a 5% CO2, 37°C incubator.

[0037] (5) When the fused cells in the wells of the culture plate have grown to 1 / 3 of the bottom of the well, the supernatant is collected and positive hybridoma cells are screened by indirect ELISA. Cells with high titer and good sensitivity are selected and transferred to a 24-well cell culture plate for expansion culture. The 24-well cell culture plate is pre-plated with feeder cells prepared with HAT medium. When the cells have grown to 1 / 2 of the bottom of the well, the supernatant is collected and the titer value is measured (Table 2).

[0038]

[0039] Five hybridoma cell lines with relatively high titers were screened and obtained, among which 4C3 had the highest titer.

[0040] Example 3: Preparation and purification of monoclonal antibodies in large quantities (1) Sterile liquid paraffin was injected intraperitoneally into parous female Bal b / c mice. Five positive hybridoma cells were cultured separately. When the cells were in the logarithmic growth phase, the positive hybridoma cells 4C3 prepared in Example 2 were diluted and injected intraperitoneally. About 7-10 days later, the abdomen of the mouse was significantly enlarged and felt tight when touched. Ascites was then collected.

[0041] (2) The preserved ascites was centrifuged and the supernatant was collected. The solution was diluted 5 times with acetic acid-sodium acetate buffer and the pH value was adjusted to 4.5. The solution was stirred at room temperature for 30 minutes. During this period, octanoic acid was slowly added dropwise to a concentration of 25µL / mL. The solution was centrifuged at 6000rpm for 30 minutes. The supernatant was collected and 1 / 10 volume of 10×PBS was added. The solution was mixed evenly and the pH value was adjusted to 7.4. The solution was cooled to 4°C and solid ammonium sulfate was added to a concentration of 0.2778g / mL. The solution was centrifuged at 6000rpm for 20 minutes and the supernatant was discarded. The precipitate was resuspended in PBS and dialyzed at 4°C for 12 hours, during which the solution was changed 2-3 times. The dialysate (purified antibody) was collected and stored at -20°C for future use. The purified antibody from 4C3 was detected by electrophoresis. The results are as follows. Figure 1 As shown, the purified antibody was a single band before reduction, and contained two specific bands after reduction, which were consistent with the sizes of the heavy chain and light chain of IgG, indicating that this method can be used to well purify antibodies secreted from 4C3 hybridoma cells, and was named monoclonal antibody 4C3.

[0042] Example 4, identification of monoclonal antibodies: (1) The purified anti-α-synuclein oligomer monoclonal antibody was assayed for binding to the α-synuclein oligomer antigen using enzyme-linked immunosorbent assay to verify the biological activity of the monoclonal antibody. The specific procedures are as follows: α-synuclein oligomer antigen was diluted to 1 μg / mL in carbonate buffer (CB, pH 9.6) and added to a 96-well microplate at a rate of 100 μL / well. The plate was incubated at 4°C overnight. The liquid was discarded and 200 μL of blocking solution (0.02 M PBS, 1% BSA, 5% sucrose) was added to each well. The plate was incubated at 37°C for 2 hours. The liquid was discarded and the plate was air-dried to obtain the antigen-coated plate.

[0043] Goat anti-mouse secondary antibody labeled with HRP: A modified sodium periodate method was used. Dissolve 5 mg of HRP in 0.5 mL of purified water. Add 0.5 mL of freshly prepared 0.1 mol / L NaIO4 and incubate at 4°C for 30 minutes, at which point the solution changes from its original brown color to dark green. Add 0.5 mL of 2.5% ethylene glycol and incubate in the dark for 30 minutes at room temperature to terminate the reaction. Add 5 mg of the antibody to be labeled and adjust the pH to 9.0 with 1.0 mol / L carbonate buffer (CB), pH 9.5. Mix thoroughly and incubate at 4°C overnight. Add 0.2 mL of 5 mg / mL sodium borohydride solution, mix thoroughly, and incubate at 4°C for 2 hours. Dialyze the solution against 0.01 mol / L, pH 7.4 PBS overnight at 4°C, changing the buffer three times. Centrifuge at 8000 rpm for 10 minutes to remove the precipitate. Collect the supernatant to obtain the HRP-labeled secondary antibody.

[0044] Add different concentrations of anti-α-synuclein oligomer monoclonal antibody to the antigen-coated plate, incubate at 37°C for 1 hour, and wash three times. Add 100 μL of a 1:5000 dilution of HRP-conjugated secondary antibody, incubate at 37°C for 1 hour, and wash three times. Add 100 μL of TMB colorimetric solution, incubate at 37°C for 15 minutes, add 50 μL of stop solution, and measure absorbance at 450 nm in a microplate reader.

[0045] See the results Figure 2 As the concentration of anti-α-synuclein oligomer monoclonal antibody increased, the OD value gradually increased. The titer of anti-α-synuclein oligomer monoclonal antibody was 0.12 ng / ml.

[0046] (2) The specificity of the anti-α-synuclein oligomer monoclonal antibody was verified by western blot.

[0047] (a) Electrophoresis gel preparation Prepare 10% separating gel and 5% stacking gel according to Tables 3 and 4.

[0048]

[0049]

[0050] (b) Sample electrophoresis Mix 40 μl of protein with 10 μl of 5× loading buffer to prepare the loading solution. Add the protein marker and protein sample sequentially, adding 20 μl of sample solution to each well. Fill the well with running buffer, close the tank lid, and connect the power supply. Initially, run the electrophoresis at 70 V constant voltage for approximately 30 minutes. Once the indicator bromophenol blue has entered the separating gel, switch to 90 V constant voltage. When the indicator reaches approximately 0.5 cm from the bottom of the gel, turn off the power supply and remove the gel plate.

[0051] (c) Transfer Moisten the PVDF membrane with anhydrous methanol for at least 30 seconds, then rinse with ddH2O for 2 minutes and soak in transfer buffer for 5 minutes. Assemble the transfer "sandwich" in the following order: black side (negative electrode) → sponge pad → 3 layers of filter paper → gel → transfer membrane → 3 layers of filter paper → sponge pad → red side (positive electrode), removing any air bubbles between the layers. Then, transfer the sandwich to the electrophoresis tank, black side facing black side. Connect the positive and negative electrodes and place the transfer cassette into the electrophoresis instrument, membrane facing the positive electrode. Add transfer buffer and place the electrophoresis instrument in ice water. Transfer the membrane at a constant current of 200 mA for 90 minutes. After transfer, quickly remove the PVDF membrane and block it in 5% BSA at room temperature for 2 hours. Wash the membrane three times with TBST for 5 minutes.

[0052] (d) Closed Prepare blocking solution: Add 2.5g of skim milk powder to 50ml of freshly prepared TBST solution and shake well. Pour some TBST solution onto a cutting plate, place the PVDF membrane on the cutting plate, mark it, and cut the membrane. Add blocking solution and incubate on a horizontal shaker at room temperature for 60 minutes. Remove the blocked membrane and rinse the hybridization membrane three times with wash solution.

[0053] (e) Antibody incubation After blocking, add the primary antibody to the hybridized membrane and incubate overnight at 4°C. Wash the membrane three times with 1× TBST (10 min each). Add the secondary antibody and incubate at 4°C for 2 h.

[0054] (f) Development Remove the membrane from the TBST buffer, remove any excess buffer, and lay it flat on a piece of cardboard, protein-side up. Add the prepared working diluent. Cover the membrane. Incubate the membrane with the working diluent for 1-5 minutes, ensuring full coverage. Remove any excess liquid, wrap the PVDF membrane in plastic wrap, and develop and fix the image with X-ray film in the dark.

[0055] like Figure 3 Channel 1 displays α-synuclein monomer antigen, Channel 2 displays α-synuclein oligomer antigen, and Channel 3 displays blood samples from PD patients. The anti-α-synuclein oligomer monoclonal antibody can recognize both α-synuclein oligomer antigen and α-synuclein monomer. Furthermore, the results in Channel 3 demonstrate that this antibody can specifically recognize α-synuclein oligomers in human blood samples.

[0056] Example 5. Acquisition of humanized variable region genes of monoclonal antibody 4C3:

[0057] (1) Total RNA of hybridoma cell 4C3 was extracted by Trizol method and cDNA was synthesized using BD SMART™ reverse transcription kit.

[0058] (2) The amplification primer (5'-CTCAGGGAARTARCCYTTGAC-3') was designed based on the mouse antibody heavy chain constant region sequence and the linker primer in the kit was used for PCR amplification to obtain the anti-α-synuclein oligomer mouse monoclonal antibody heavy chain fragment, which was sequenced after the pGEM-T vector was constructed.

[0059] (3) The amplification primer (5'-TCACTGCCATCAATCTTCCAC-3') was designed based on the mouse antibody light chain constant region sequence and the adapter primer in the kit was used for PCR amplification to obtain the anti-α-synuclein oligomer mouse monoclonal antibody light chain fragment, which was sequenced after the pGEM-T vector was constructed.

[0060] (4) CDR analysis of the anti-α-synuclein oligomer mouse monoclonal antibody 4C3.

[0061] Based on the sequenced heavy and light chain variable region sequences of the 4C3 antibody, and referring to the antibody CDR analysis definition method at http: / / www.bioinf.org.uk / abs / #cdrdef, the CDR sequence of the light chain variable region of the anti-oligomer mouse monoclonal antibody 4C3 was obtained, as shown in Table 5. The CDR sequence of the heavy chain variable region of the anti-α-synuclein oligomer mouse monoclonal antibody 4C3 was obtained, as shown in Table 6.

[0062]

[0063]

[0064] (5) Obtaining the sequence of humanized monoclonal antibody 4C3 The obtained framework regions of the heavy chain variable region (HFR1, HFR2, HFR3, HFR4) and the light chain variable region (LFR1, LFR2, LFR3, LFR4) of the anti-α-synuclein oligomer mouse monoclonal antibody 4C3 were aligned using the IgBlast search database (IMGT human Vgenes (F+ORF+in-frameP)) to obtain the human FR region with the highest homology. The domain amino acids of the human framework region were further considered to determine the human framework region sequence, which was combined with the light chain variable region CDR and heavy chain variable region CDR of the anti-α-synuclein oligomer mouse monoclonal antibody 4C3 to obtain the following: The light chain variable region of the humanized anti-α-synuclein oligomer monoclonal antibody H4C3 is shown in SEQ ID NO: 7.

[0065] The heavy chain variable region of the humanized anti-α-synuclein oligomer monoclonal antibody H4C3 is shown in SEQ ID NO: 8.

[0066] The light chain variable region and heavy chain variable region of the murine anti-α-synuclein oligomer mouse monoclonal antibody 4C3 differ from those of the humanized anti-α-synuclein oligomer mouse monoclonal antibody H4C3 only in the murine framework regions, while the CDRs remain unchanged. The framework regions in the humanized antibody can be replaced with murine ones according to conventional techniques to obtain the light chain variable region and heavy chain variable region of the murine anti-α-synuclein oligomer mouse monoclonal antibody 4C3.

[0067] Example 6: Eukaryotic expression of humanized anti-α-synuclein oligomer mouse monoclonal antibody H4C3: 1. Preparation of humanized anti-α-synuclein oligomer mouse monoclonal antibody H4C3.

[0068] 1. Construction of eukaryotic expression vector The variable regions of the humanized anti-α-synuclein oligomer mouse monoclonal antibody H4C3 were linked to the human IgG constant region (Fc) gene to construct full-length heavy and light chain genes. These were then recombined into the eukaryotic expression vector pcDNA3.1 using the corresponding restriction sites.

[0069] The details are as follows: The vector for expressing the antibody H4C3 light chain is obtained by replacing a DNA molecule composed of the humanized anti-α-synuclein oligomer mouse monoclonal antibody H4C3 light chain variable region and the human IgG light chain constant region gene between the BamHI and EcoRI restriction sites of the eukaryotic expression vector pcDNA3.1; the vector expresses the monoclonal antibody H4C3 light chain; The nucleotide sequence of the DNA molecule consisting of the humanized anti-α-synuclein oligomer mouse monoclonal antibody H4C3 light chain variable region and the human light chain constant region gene consists of SEQ ID NO: 19 and SEQ ID NO: 21 (human IgG light chain constant region gene); The amino acid sequence of the light chain of the monoclonal antibody H4C3 consists of SEQ ID NO: 7 and SEQ ID NO: 9 (human IgG light chain constant region).

[0070] The vector for expressing the heavy chain of the antibody H4C3 is obtained by replacing the DNA molecule composed of the humanized anti-α-synuclein oligomer mouse monoclonal antibody H4C3 heavy chain variable region and the human IgG heavy chain constant region gene between the BamHI and EcoRI restriction sites of the eukaryotic expression vector pcDNA3.1; the vector expresses the heavy chain of the monoclonal antibody H4C3; The nucleotide sequence of the DNA molecule consisting of the humanized anti-α-synuclein oligomer mouse monoclonal antibody H4C3 heavy chain variable region and the human IgG heavy chain constant region gene consists of SEQ ID NO: 20 and SEQ ID NO: 23 (human IgG heavy chain constant region gene); The amino acid sequence of the heavy chain of the monoclonal antibody H4C3 consists of SEQ ID NO: 8 and SEQ ID NO: 11 (human IgG heavy chain constant region).

[0071] 2. Eukaryotic expression of antibodies The vector expressing the antibody H4C3 light chain and the vector expressing the antibody H4C3 heavy chain prepared in 1 above were linearized and digested with EcoRI enzyme, respectively. The linearized vector expressing the antibody H4C3 light chain and the linearized vector expressing the antibody H4C3 heavy chain were then electroporated together at 300 V and 900 μF using a BioRad Xcell electroporator to transfect CHO-K1-38 cells (Nanjing GenScript Biotechnology Co., Ltd., M00553). 48 hours after transfection, the culture medium was replaced with antibiotic-containing culture medium. The medium was changed every 2 days for 2 weeks, and the culture medium was collected.

[0072] The culture medium was centrifuged to collect the cell culture supernatant, and the antibodies in the supernatant were detected by indirect ELISA: the collected cell supernatant was added to the microplate coated with α-synuclein oligomer antigen, incubated at 37°C for 1 hour, and the HRP-labeled secondary antibody was added after washing. The plate was incubated at 37°C for 1 hour, and the substrate TMB was added for color development after washing. The reaction was terminated by adding sulfuric acid, and the absorbance was measured using a microplate reader (at 450 nm).

[0073] The results are shown in Table 7 below. It can be seen that the supernatants of cells with different numbers all contain 4C3 monoclonal antibody.

[0074]

[0075] 3. Purification of humanized anti-α-synuclein oligomer monoclonal antibody H4C3.

[0076] Affinity purification was performed using Beaver Bio Magrose Protein A antibody purification magnetic beads.

[0077] (1) Magnetic bead pretreatment: Vortex the antibody purification magnetic beads for 30 seconds to fully resuspend them; take 10% (v / v) of the magnetic bead suspension and place it in another new 1.5 mL centrifuge tube. Perform magnetic separation on the magnetic bead suspension, discard the supernatant, wash twice with Binding / Washing buffer, and perform magnetic separation. The magnetic beads in the tube can be directly used for protein separation.

[0078] Note: The amount of magnetic beads used in this step can be adjusted based on the maximum binding capacity of the target protein. When the concentration of the target protein is greater than 150µg / mL, 1.2-1.5 times the amount of magnetic beads can be used. If the concentration of the target protein is too low, such as below 70µg / mL, the amount of magnetic beads can be increased to improve protein recovery, for example, up to 3 times the amount of magnetic beads.

[0079] (2) Protein adsorption: Add the magnetic beads pretreated in the above steps to 1 ml of sample solution, shake well, and place in a flip mixer to mix at 2-8°C overnight to ensure full contact and adsorption between the sample and the magnetic beads. Collect the magnetic beads by magnetic separation and discard the supernatant.

[0080] (3) Magnetic bead washing: Add 1 mL of Binding / Washing buffer to the centrifuge tube, oscillate to resuspend the magnetic beads, and then perform magnetic separation. Discard the supernatant; repeat this operation 3 times.

[0081] (4) Protein elution: Add 1 mL of elution buffer to the centrifuge tube where the magnetic beads have been washed. Quickly resuspend the beads by pipetting or vortexing. Place the tube in a flip mixer at room temperature (approximately 25°C) or gently flip the tube manually. After flipping for 10 minutes, perform magnetic separation and collect the supernatant into a new centrifuge tube.

[0082] Note: The amount of Elution buffer used in this step is recommended to control the final eluted protein concentration to 0.6-1.2 mg / mL. At this time, more than 95% of the protein in the first elution condition will be eluted. If the amount of Elution buffer used is too small, some protein will remain on the magnetic beads during the first elution, resulting in a lower protein recovery rate.

[0083] (5) Neutralization pH: Add a certain amount of Neutrilization buffer to the protein elution solution in the above step, generally 1 / 10 of the elution volume, to ultimately keep the pH value of the eluted protein in a neutral environment, so as to maintain the biological activity of the protein and avoid protein inactivation.

[0084] (6) Centrifugation: 12000 rpm, 4°C for 10 min, and collect the supernatant. The obtained product is the purified humanized anti-α-synuclein oligomer monoclonal antibody H4C3.

[0085] The purified humanized anti-α-synuclein oligomer monoclonal antibody H4C3 was sequenced, and the expression was confirmed to be H4C3 monoclonal antibody. The sequences of the light chain and heavy chain of the H4C3 monoclonal antibody were consistent with the aforementioned light chain and heavy chain sequences of H4C3, indicating that the expressed antibody was H4C3 monoclonal antibody.

[0086] 2. Biological activity detection of humanized and mouse anti-α-synuclein oligomer monoclonal antibodies H4C3 / 4C3.

[0087] The difference in binding specificity of the purified humanized anti-α-synuclein oligomer monoclonal antibody H4C3 obtained above to the α-synuclein oligomer antigen was determined by enzyme-linked immunosorbent assay to verify the biological activity of the humanized monoclonal antibody. The specific operation is as follows: (1) Dilute the α-synuclein oligomer antigen to 1 μg / mL with sodium bicarbonate buffer (pH 9.5), coat the ELISA plate, block, wash, and air dry for later use; (2) Add 100 μL of the purified humanized anti-α-synuclein oligomer monoclonal antibody H4C3 obtained above (diluted to 2 μg / ml in a PBS buffer with a pH of 7.4 and a concentration of 0.02 mol / L), incubate, and wash; (3) Add 100 μL of HRP-labeled goat anti-human IgG antibody, incubate, and wash; (4) After adding the substrate TMB for color development, sulfuric acid is added to terminate the reaction and the absorbance is measured on a microplate reader (at 450 nm).

[0088] (5) Mouse monoclonal antibody 4C3 and HRP-labeled goat anti-mouse IgG were used as controls to compare the differences in the measurement values of the two antibodies.

[0089] Test results see Figure 4 It can be seen that the humanized monoclonal antibody H4C3 has the same specificity for α-synuclein oligomer antigen as the mouse monoclonal antibody 4C3, indicating that the humanized monoclonal antibody maintains the biological activity against α-synuclein oligomer antigen.

[0090] Example 7, Preparation and Performance Verification of α-Synuclein Oligomer Detection Kit: 1. Preparation of the kit The H4C3 antibody is used to prepare a magnetic microparticle chemiluminescence kit, which includes: H4C3 antibody magnetic beads, enzyme conjugate, calibrator / quality control product, washing solution and luminescent solution.

[0091] (1) Preparation of H4C3 antibody magnetic beads: Take 10 mg of magnetic beads, add 1 ml of MES buffer (0.1 M, pH 5.0), wash once, then add 10 μl of 10 mg / ml EDC for activation, activate at room temperature for 30 minutes, 0.2 mg of the humanized α-synuclein oligomer antibody H4C3 prepared in Example 6 was added, mixed and reacted at room temperature for 2 hours, 1 ml of 1% BSA was added, blocked at room temperature for 2 hours, washed three times with PBS containing 0.01% Tween 20, and finally added to 1 ml of PBS containing 1% BSA for storage.

[0092] (2) Preparation of enzyme conjugates: SMCC method was used.

[0093] A. To 1 mg of ALP solution, add 39 μl of 1 mg / ml Sulfo-SMCC at a molar ratio of 5:1. Incubate at 25°C for 30 min. Remove unreacted SMCC by ultrafiltration using 0.02 M PBS. Ultrafiltration is performed three times. Centrifuge at 4000 rpm for 5 min at 20°C.

[0094] B. Take 1 mg of IgG solution, add 4 μl of 1 M DTT, and react at 25°C for 30 minutes. Remove unreacted DTT by ultrafiltration using 0.02 M PBS as the buffer. Ultrafiltration is performed three times, and centrifugation is performed at 4000 rpm for 5 minutes at 20°C.

[0095] C. Combine the collected products from steps A and B, react at 25°C for 1 hour, add 10 μL of 1 mol / L 2-mercaptoethanol, and react at 25°C for 30 minutes. Remove unreacted βME by ultrafiltration using 0.02 M PBS as the buffer. Ultrafiltration is repeated three times, followed by centrifugation at 4000 rpm for 5 minutes at 20°C. Add an equal volume of glycerol.

[0096] (3) Preparation of calibrators / quality control products: The calibrator diluent was PBS buffer (pH 7.4, 0.02 M) containing 1% BSA and 0.02% (volume percentage) Proclin-300, serving as the zero calibrator (S0).

[0097] Calibrator solutions S1 to S5 contain different concentrations of α-synuclein oligomer antigen diluted in calibrator diluent. The concentrations are 12.5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL, respectively.

[0098] The quality control solutions QC1 and QC2 are solutions containing different concentrations of α-synuclein oligomer antigen diluted with calibrator diluent. The concentrations are 25 ng / mL and 100 ng / mL, respectively.

[0099] (4) Preparation of washing solution: Weigh 2.9 g disodium hydrogen phosphate dodecahydrate, 0.3 g sodium dihydrogen phosphate dihydrate, 8.5 g sodium chloride, and 0.5 ml Tween 20, and add purified water to make up to 1 L.

[0100] (5) Luminescent liquid was purchased from Wuhan Hanhai New Enzyme Co., Ltd., product number HH3605.

[0101] The above components are assembled into a kit.

[0102] 2. How to use the kit The α-synuclein oligomer detection kit prepared above is used in conjunction with a recent fully automatic chemiluminescence analyzer. The detection procedure is as follows: (1) Pipette 50 μl of antibody magnetic beads, apply magnetic field to adsorb the magnetic beads, and remove the magnetic bead storage solution.

[0103] (2) Pipette 50 μl of sample, mix with magnetic beads and incubate at 37°C for 15 min.

[0104] (3) Apply a magnetic field to adsorb the magnetic beads, remove the sample solution after the reaction, and wash the magnetic beads twice with 200 μl of washing solution.

[0105] (4) Pipette 100 μl of enzyme conjugate, mix with magnetic beads and incubate at 37°C for 15 min.

[0106] (5) Apply a magnetic field to adsorb the magnetic beads, remove the enzyme after the reaction, and wash the magnetic beads twice with 200 μl of washing solution.

[0107] (6) Pipette 100 μl of luminescent solution, mix with magnetic beads, incubate at 37°C for 3 min, and detect the luminescence value.

[0108] 3. Detection of specific recognition of α-synuclein oligomers The α-synuclein polypeptide monomer and α-synuclein oligomer antigen were diluted with diluent to form a series of gradients with equal molar concentrations, and the above-prepared kit was used in conjunction with a nearly automatic chemiluminescence instrument for detection. The results were as follows: Figure 5 .

[0109] From above Figure 5 It can be seen that with the increase in the molar concentration of the antigen, the detection signal of the α-synuclein oligomer antigen increases gradually, but the detection signal of the α-synuclein monomer remains low, indicating that the kit established by the present invention can specifically recognize α-synuclein oligomers but not monomers.

[0110] 4. Performance indicators of the test kit (blank limit, repeatability, specificity) (1) Blank limit Use the kit prepared as described above and employ a zero-concentration calibrator as a sample for detection. Repeat the measurement 20 times to obtain the absorbance values (A values) of the 20 measurement results. Calculate the mean (M) and standard deviation (SD) to obtain the A value corresponding to M+2SD. Based on the dose-response curve of the calibrator used in the kit, substitute the A value corresponding to M+2SD into the curve to determine the corresponding concentration value, which is the blank limit.

[0111] (2) Repeatability The quality control products (Qc1, Qc2) were tested using the kit prepared above, each test was repeated 10 times, and the mean concentration (Mean) and standard deviation (SD) of the test results were calculated.

[0112] Coefficient of variation (CV) = SD / Mean×100%.

[0113] (3) Specificity A 500 ng / ml α-synuclein monomer solution was used as a specific sample and the assay was performed using the kit prepared above. The assay was repeated twice, and the average value (M) of the results was calculated.

[0114] Cross-reaction rate = M / 500 × 100%.

[0115] The above performance index results are shown in Table 8.

[0116]

[0117] Example 8: α-synuclein oligomer detection kit for measuring the α-synuclein oligomer content in the blood of normal people and PD patients:

[0118] EDTA-anticoagulated blood samples were collected from a hospital, including 50 healthy controls and 50 samples from patients with a clinical diagnosis of Parkinson's disease (PD). After centrifugation, the red blood cells (RBCs) were removed and aliquoted and stored at -80°C for later use.

[0119] After thawing at room temperature, the frozen red blood cells were diluted 20-fold with 0.02M PBS, shaken and mixed, and tested using the detection kit and detection procedure prepared in Example 7. The results are shown in Tables 9-11 below:

[0120] From Table 9-11 and Figure 6 As can be seen, there is a significant difference between the normal group and the PD group (P < 0.01), and the test results can distinguish the two groups of samples. The kit prepared using this method can be used for PD screening.

[0121] Example 9, comparison of α-synuclein oligomer content in blood and saliva measured by α-synuclein oligomer detection kit: EDTA anticoagulated blood and saliva samples were collected from a hospital, including 30 normal controls (numbered 1-30) and 20 samples (numbered 31-50) from patients with a clinical diagnosis of Parkinson's disease (PD). The anticoagulated blood was centrifuged to separate the plasma, and the saliva was centrifuged to obtain the supernatant. The test kit and procedure prepared in Example 7 were used for testing. The results are shown in Tables 12 and 12. Figure 7 :

[0122] Figure 7 The results showed that the correlation between the detection results of the two sample types was r>0.95 (r=0.9617), which was a good correlation. The reagent prepared by the present invention can be applied to the detection of various sample types.

[0123] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A humanized monoclonal antibody for detecting human α-synuclein oligomers, characterized in that The light chain CDR sequence of the binding portion includes at least one of LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 1; the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:

3.

2. The humanized monoclonal antibody for detecting human α-synuclein oligomers according to claim 1, characterized in that The heavy chain CDR sequence of the binding portion includes at least one of HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 4; the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:

6.

3. The humanized monoclonal antibody for detecting human α-synuclein oligomers according to claim 1, characterized in that The preparation method of the human α-synuclein oligomer is as follows: diluting the human α-synuclein polypeptide with a PBS buffer solution, incubating at a constant temperature with shaking, then adding sodium dodecyl sulfate, sonicating, centrifuging, collecting the supernatant, and freeze-drying to obtain the human α-synuclein oligomer.

4. The humanized monoclonal antibody for detecting human α-synuclein oligomers according to claim 3, characterized in that The constant temperature shaking incubation temperature is 36-38° C., the shaking frequency is 500-1000 rpm, and the time is 60-80 h.

5. The humanized monoclonal antibody for detecting human α-synuclein oligomers according to claim 3, characterized in that The ultrasonic time is 20-40 minutes, and the centrifugal temperature is 2-5° C. and the time is 20-40 minutes.

6. The humanized monoclonal antibody for detecting human α-synuclein oligomers according to claim 3, characterized in that The pH value of the PBS buffer is 7-7.7, the dilution concentration is 3-7 mg / mL, and the final concentration of the sodium lauryl sulfate is 2-4 wt %.

7. A method for specifically detecting α-synuclein oligomers using the humanized monoclonal antibody for detecting human α-synuclein oligomers according to any one of claims 1 to 6, characterized in that: include: The antibodies are respectively combined with a solid phase carrier and a marker to prepare solid phase antibodies and labeled antibodies, which are then added to the sample for incubation to generate a signal.

8. A detection kit prepared using the humanized monoclonal antibody for detecting human α-synuclein oligomers according to any one of claims 1 to 6 as a raw material.

9. Use of the detection kit according to claim 8 in auxiliary diagnosis of Parkinson's disease.

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