An antibody binding to Aβ oligomers and its application

Abstract

The present invention provides an antibody that binds to Aβ oligomers and its application, belonging to the field of medical technology. The antibody comprises: an L chain having CDR1, CDR2, and CDR3, wherein the amino acid sequence of the L chain of CDR1 is as shown in SEQ ID NO.1; the amino acid sequence of the L chain of CDR2 is as shown in SEQ ID NO.2; and the amino acid sequence of the L chain of CDR3 is as shown in SEQ ID NO.3; and an H chain having CDR1, CDR2, and CDR3, wherein the amino acid sequence of the H chain of CDR1 is as shown in SEQ ID NO.4; and the amino acid sequence of the H chain of CDR2 is as shown in SEQ ID NO.5. The present invention provides a monoclonal antibody that can bind to Aβ oligomers, as well as a method for preparing the monoclonal antibody and its application. Using the monoclonal antibody as a raw material and a unique repeating epitope sandwich method, Aβ oligomers can be detected with high specificity, high sensitivity, and high accuracy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an antibody binding to Aβ oligomers and applications thereof. Background Art

[0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system that occurs in the elderly and pre-elderly, characterized by progressive cognitive dysfunction and behavioral impairment. Clinically, it manifests as memory impairment, aphasia, apraxia, agnosia, impairment of visual-spatial abilities, impairment of abstract thinking and calculation, and personality and behavioral changes. AD is the most common type of dementia in the elderly, accounting for 60-70% of cases. The risk of developing the disease increases with age. As the global aging process accelerates, the number of AD patients continues to increase, placing a heavy burden on individuals, families, and society.

[0003] Amyloidosis occurs in the brain and is the primary characteristic of Alzheimer's disease. Many experts believe that amyloid plaques (Aβ; primarily Aβ1-40 and Aβ oligomers) are closely linked to the pathophysiology of AD. Recent studies suggest that soluble aggregates of amyloid β oligomers (AβO), rather than amyloid fibrils, are more relevant to the pathophysiology of AD.

[0004] Compared to monomers and fibrils, AβO is the most neurotoxic, damaging neurons and synapses, leading to memory loss and a higher correlation with disease than other markers. Blood AβO levels are elevated in the early stages of AD, including subjective cognitive decline (SCD) and mild cognitive impairment (MCI), before the onset of AD. Tracking AβO detection at the onset of AD and at different stages during its development is crucial for early detection, diagnosis, and disease progression monitoring. Summary of the Invention

[0005] The purpose of the present invention is to provide an antibody that binds to Aβ oligomers and its application. Using the monoclonal antibody as raw material and a unique repeat epitope sandwich method, Aβ oligomers can be detected with high specificity, high sensitivity and high accuracy.

[0006] The technical solution of the present invention is achieved as follows:

[0007] The present invention provides an antibody that binds to Aβ oligomers, the antibody comprising:

[0008] An L chain having CDR1, CDR2, and CDR3, wherein the amino acid sequence of the L chain of CDR1 is as shown in SEQ ID NO.1; the amino acid sequence of the L chain of CDR2 is as shown in SEQ ID NO.2; and the amino acid sequence of the L chain of CDR3 is as shown in SEQ ID NO.3;

[0009] An H chain having CDR1, CDR2, and CDR3, wherein the amino acid sequence of the H chain of CDR1 is such as SEQ ID NO.4; the amino acid sequence of the H chain of CDR2 is such as SEQ ID NO.5; and the amino acid sequence of the H chain of CDR3 is such as SEQ ID NO.6.

[0010] As a further improvement of the present invention, the preparation method of the Aβ oligomer is as follows: Aβ polypeptide is dissolved in DMSO, diluted with PBS, incubated with constant temperature shaking, centrifuged, supernatant is collected, and freeze-dried to obtain Aβ oligomer.

[0011] As a further improvement of the present invention, the pH value of the PBS solution is 7.4, and the dilution is to 1.5-2.5 mg / mL.

[0012] As a further improvement of the present invention, the constant temperature oscillation incubation temperature is 35-40° C., the time is 44-52 h, and the oscillation frequency is 500-1000 rpm.

[0013] The present invention further protects the use of the above-mentioned antibody binding to Aβ oligomers in the preparation of AD auxiliary diagnosis products.

[0014] The present invention further protects a detection kit using the above-mentioned antibody binding to Aβ oligomers as a raw material.

[0015] The present invention further protects the use of the above-mentioned detection kit in the preparation of AD auxiliary diagnosis products.

[0016] The present invention has the following beneficial effects:

[0017] The present invention provides a monoclonal antibody that can bind to Aβ oligomers, as well as a preparation method and application of the monoclonal antibody. Using the monoclonal antibody as raw material and a unique repeating epitope sandwich method, Aβ oligomers can be detected with high specificity, high sensitivity, and high accuracy.

[0018] The present invention uses Aβ polypeptide monomers as raw materials and oscillates at a constant temperature of 37°C to aggregate them. The prepared Aβ oligomers are used as immunogens to immunize mice. Mouse spleen cells are collected to prepare hybridoma cells. Cells that can secrete and specifically recognize Aβ oligomers are screened by indirect ELISA. Monoclonal antibodies prepared and purified from the cell lines are used for the detection of Aβ oligomers, which can be used for auxiliary diagnosis of Alzheimer's disease.

[0019] The present invention also provides a kit for detecting the content of Aβ oligomers in human blood, which can be used for auxiliary diagnosis of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 This is the electrophoresis diagram of monoclonal antibody AC1-4;

[0022] Figure 2 is a titer graph of anti-Aβ oligomer monoclonal antibodies;

[0023] Figure 3 This is the Western result of monoclonal antibody AC1-4;

[0024] Figure 4 This is a comparison chart of antibody activity;

[0025] Figure 5 is the assay specificity map;

[0026] Figure 6 This is a comparison of plasma Aβ oligomer levels between AD and normal mice;

[0027] Figure 7 Comparison of Aβ oligomer content in brain tissues of AD and normal mice;

[0028] Figure 8 A comparison chart between normal people and AD patients. DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1. Preparation of Aβ oligomers

[0031] 1. Dissolve 5 mg of Aβ42 peptide (purchased from Hubei Qiangyao Biotechnology) in hexafluoroisopropanol (HFIP) to make a 1 mg / ml solution. Block the solution at room temperature for 60 minutes, place it on ice for 10 minutes, and then place it in a fume hood with the lid open and evaporate it overnight at room temperature to allow the HFIP to evaporate completely.

[0032] 2. Add a small amount of dimethyl sulfoxide (DMSO) to the treated polypeptide tube to re-dissolve it, dilute it with PBS to 2 mg / ml, and then place it on a constant temperature shaker at 37 degrees and set the shaking frequency to 800 rpm.

[0033] 3. After shaking for 48 hours, remove the tube and centrifuge at 10,000 rpm for 30 minutes at 4 degrees. The supernatant is Aβ oligomers.

[0034] 1. Animal immunization and polyclonal antibody titer determination

[0035] (1) Animal selection: Five healthy female Balb / c mice aged 5 weeks were selected and immunized with the immunogen prepared in Example 1.

[0036] (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.

[0037] (3) Immunization dose: 0.1 mg / animal / time.

[0038] (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.

[0039] (5) Aβ oligomers were coated on an enzyme-linked immunosorbent assay plate and then blocked. Polyclonal antibody serum and enzyme-labeled secondary antibody were added and a color reaction was performed to determine the titer of the polyclonal antibody. The results are shown in Table 1.

[0040] Table 1: Determination of mouse polyclonal antibody titer

[0041]

[0042] Judging from the preliminary ELISA test results, mice numbered 3 had the best immune effect.

[0043] 2. Cell fusion and hybridoma cell screening.

[0044] (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.

[0045] (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.

[0046] (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.

[0047] (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.

[0048] (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).

[0049] Table 2: Titers of screened monoclonal antibodies

[0050]

[0051] Five hybridoma cell lines with relatively high titers were screened and obtained, among which AC1-4 had the highest titer.

[0052] (1) Sterile liquid paraffin was injected into the peritoneal cavity of multiparous 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 AC1-4 prepared in Example 2 were diluted and injected into the peritoneal cavity. About 7-10 days later, the abdomen of the mouse was significantly enlarged and felt tight when touched. Ascites was then collected.

[0053] (2) The preserved ascites was centrifuged and the supernatant was collected. The supernatant was diluted 5-fold with acetic acid-sodium acetate buffer and adjusted to pH 4.5. The solution was stirred at room temperature for 30 min. During this time, octanoic acid was slowly added dropwise to a concentration of 25 μL / mL. The solution was centrifuged at 6000 rpm for 30 min. The supernatant was collected and 1 / 10 volume of 10× PBS was added. The solution was mixed evenly and the pH was adjusted to 7.4. The solution was cooled to 4°C and solid ammonium sulfate was added to a concentration of 0.2778 g / mL. The solution was centrifuged at 6000 rpm for 20 min. The supernatant was discarded. The pellet was resuspended in PBS and dialyzed at 4°C for 12 h, with the solution changed 2-3 times. The dialysate (purified antibody) was collected and stored at -20°C for later use.

[0054] The purified antibodies from AC1-4 were detected by electrophoresis. 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 AC1-4 hybridoma cells, and was named monoclonal antibody AC1-4.

[0055] (1) The purified anti-Aβ oligomer monoclonal antibody was tested for its binding to the Aβ oligomer antigen using enzyme-linked immunosorbent assay to verify the biological activity of the monoclonal antibody. The specific procedures are as follows:

[0056] Aβ 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 allowed to dry to obtain the antigen-coated plate.

[0057] 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.

[0058] Add different concentrations of anti-Aβ 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.

[0059] See the results Figure 2 As the concentration of anti-Aβ oligomer monoclonal antibody increased, the OD value gradually increased. The titer of anti-Aβ oligomer monoclonal antibody was 0.12 ng / ml.

[0060] (2) The specificity of anti-Aβ oligomer monoclonal antibodies was verified by western blot.

[0061] a) Electrophoresis gel preparation

[0062] Prepare 10% separating gel and 5% stacking gel according to Tables 3 and 4.

[0063] Table 3: 10% separation gel formula

[0064]

[0065] Table 4: 5% stacking gel formula

[0066]

[0067] b) Sample electrophoresis

[0068] 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.

[0069] C) Transfer

[0070] 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.

[0071] d) Closed

[0072] 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.

[0073] e) Antibody incubation

[0074] 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.

[0075] f) Development

[0076] 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.

[0077] like Figure 3Channel 1 shows Aβ oligomer antigens, Channel 2 shows Aβ monomer antigens, and Channel 3 shows blood samples from AD patients. The anti-Aβ oligomer monoclonal antibody can recognize both Aβ oligomer antigens and Aβ monomers. Furthermore, the results in Channel 3 demonstrate that this antibody can specifically recognize Aβ oligomers in human blood samples.

[0078] 1) Total RNA from hybridoma cells AC1-4 was extracted using the Trizol method and cDNA was synthesized using the BD SMART™ reverse transcription kit.

[0079] 2) PCR amplification was performed using a primer designed based on the mouse antibody heavy chain constant region sequence (5'-CTCAGGGAARTARCCYTTGAC-3') and the adapter primer provided in the kit to obtain the anti-Aβ oligomer mouse monoclonal antibody heavy chain fragment. The fragment was then sequenced after the pGEM-T vector was constructed.

[0080] 3) PCR amplification was performed using a primer designed based on the mouse antibody light chain constant region sequence (5'-TCACTGCCATCAATCTTCCAC-3') and the adapter primer provided in the kit to obtain the anti-Aβ oligomer mouse monoclonal antibody light chain fragment. The fragment was then sequenced after the pGEM-T vector was constructed.

[0081] 4) CDR analysis of anti-Aβ oligomer mouse monoclonal antibody AC1-4.

[0082] Based on the sequenced heavy and light chain variable region sequences of the AC1-4 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 AC1-4 was obtained, as shown in Table 5. The CDR sequence of the heavy chain variable region of the anti-Aβ oligomer mouse monoclonal antibody AC1-4 was obtained, as shown in Table 6.

[0083] Table 5: CDR sequences of the light chain variable region of anti-Aβ oligomer mouse monoclonal antibody AC1-4

[0084]

[0085] Table 6: CDR sequences of the heavy chain variable region of anti-Aβ oligomer murine monoclonal antibody AC1-4

[0086]

[0087] 5) Obtain the sequence of humanized monoclonal antibody AC1-4

[0088] 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-Aβ oligomer mouse monoclonal antibody AC1-4 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-Aβ oligomer mouse monoclonal antibody AC1-4, respectively, to obtain the following:

[0089] The light chain variable region of the humanized anti-Aβ oligomer monoclonal antibody HAC1-4 is shown in SEQ ID NO: 7.

[0090] The heavy chain variable region of the humanized anti-Aβ oligomer monoclonal antibody HAC1-4 is shown in SEQ ID NO: 8.

[0091] The light chain variable region and heavy chain variable region of the murine anti-Aβ oligomer mouse monoclonal antibody AC1-4 differ from the humanized anti-Aβ oligomer monoclonal antibody HAC1-4 only in the murine framework region, while the CDR remains unchanged. The framework region in the humanized antibody can be replaced with the murine one according to conventional techniques to obtain the light chain variable region and heavy chain variable region of the murine anti-Aβ oligomer mouse monoclonal antibody AC1-4.

[0092] 1. Preparation of humanized anti-Aβ oligomer mouse monoclonal antibody HAC1-4.

[0093] 1. Construction of eukaryotic expression vector

[0094] The variable regions of the humanized anti-Aβ oligomer mouse monoclonal antibody HAC1-4 were linked to the human IgG constant region (Fc) gene to construct the full-length heavy and light chain genes. These were then recombined into the eukaryotic expression vector pcDNA3.1 using the corresponding restriction sites.

[0095] The details are as follows:

[0096] The vector expressing the antibody HAC1-4 light chain is obtained by replacing the DNA molecule composed of the humanized anti-Aβ oligomer mouse monoclonal antibody HAC1-4 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 HAC1-4 light chain;

[0097] The nucleotide sequence of the DNA molecule consisting of the humanized anti-Aβ oligomer mouse monoclonal antibody HAC1-4 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);

[0098] The amino acid sequence of the light chain of the monoclonal antibody HAC1-4 consists of SEQ ID NO: 7 and SEQ ID NO: 9 (human IgG light chain constant region).

[0099] The vector expressing the heavy chain of the antibody HAC1-4 is obtained by replacing the DNA molecule composed of the humanized anti-Aβ oligomer mouse monoclonal antibody HAC1-4 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 HAC1-4;

[0100] The nucleotide sequence of the DNA molecule consisting of the humanized anti-Aβ oligomer murine monoclonal antibody HAC1-4 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);

[0101] The amino acid sequence of the heavy chain of the above monoclonal antibody HAC1-4 consists of SEQ ID NO: 8 and SEQ ID NO: 11 (human IgG heavy chain constant region).

[0102] 2. Eukaryotic expression of antibodies

[0103] The vector expressing the antibody HAC1-4 light chain and the vector expressing the antibody HAC1-4 heavy chain prepared in 1 above were linearized and digested with EcoRI enzyme, respectively. The linearized vector expressing the antibody HAC1-4 light chain and the linearized vector expressing the antibody HAC1-4 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 a culture medium containing antibiotics. The culture medium was replaced every 2 days for 2 weeks, and the culture medium was collected.

[0104] The culture medium was centrifuged to collect the cell culture supernatant, and the antibodies in the supernatant were detected by indirect ELISA: the cell supernatant collected above was added to the microplate coated with Aβ 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 on a microplate reader (at 450 nm).

[0105] The results are shown in Table 7 below. It can be seen that the supernatants of cells with different numbers all contain AC1-4 monoclonal antibody.

[0106] Table 7: Antibody test results

[0107]

[0108] 3. Purification of humanized anti-Aβ oligomer monoclonal antibodies HAC1-4.

[0109] Affinity purification was performed using Beaver Bio Magrose Protein A antibody purification magnetic beads.

[0110] (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.

[0111] 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.

[0112] (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.

[0113] (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.

[0114] (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.

[0115] 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.

[0116] (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.

[0117] (6) Centrifugation: 12000 rpm, 4°C for 10 min, and collect the supernatant. The obtained supernatant is the purified humanized anti-Aβ oligomer monoclonal antibody HAC1-4.

[0118] The purified humanized anti-Aβ oligomer monoclonal antibody HAC1-4 was sequenced, and the expression was confirmed to be HAC1-4 monoclonal antibody. The sequences of the light chain and heavy chain of HAC1-4 monoclonal antibody were consistent with those of the aforementioned HAC1-4, indicating that the antibody expressed was HAC1-4 monoclonal antibody.

[0119] 2. Biological activity detection of humanized and mouse anti-Aβ oligomer monoclonal antibodies HAC1-4 / AC1-4.

[0120] The enzyme-linked immunosorbent assay was used to determine the differences in the binding specificity of the purified humanized anti-Aβ oligomer monoclonal antibodies HAC1-4 obtained above to the Aβ oligomer antigen to verify the biological activity of the humanized monoclonal antibodies. The specific procedures are as follows:

[0121] 1) Dilute Aβ oligomer antigen to 1 μg / mL with sodium bicarbonate buffer (pH 9.5), coat the plate with an enzyme-linked immunosorbent assay (ELISA) plate, block the plate, wash the plate, and air dry.

[0122] 2) Add 100 μL of the purified humanized anti-Aβ oligomer monoclonal antibody HAC1-4 obtained above (diluted to 2 μg / ml in PBS buffer, pH 7.4, 0.02 mol / L), incubate, and wash;

[0123] 3) Add 100 μl of HRP-labeled goat anti-human IgG antibody, incubate, and wash;

[0124] 4) After adding the substrate TMB for color development, add sulfuric acid to stop the reaction and measure the absorbance on a microplate reader (at 450 nm).

[0125] 5) Mouse monoclonal antibody AC1-4 and HRP-labeled goat anti-mouse IgG were used as controls to compare the differences in the measured values ​​of the two antibodies.

[0126] Test results see Figure 4 It can be seen that the humanized monoclonal antibody HAC1-4 has the same specificity for Aβ oligomer antigen as the mouse monoclonal antibody AC1-4, indicating that the humanized monoclonal antibody maintains the biological activity against Aβ oligomer antigen.

[0127] 1. Preparation of the kit

[0128] A magnetic microparticle chemiluminescence kit is prepared using HAC1-4 antibodies. The components include: HAC1-4 antibody magnetic beads, enzyme conjugates, calibrators / quality controls, wash solution, and luminescent solution.

[0129] 1) Preparation of HAC1-4 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 at room temperature for 30 minutes.

[0130] 0.2 mg of the humanized Aβ oligomer antibody HAC1-4 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.

[0131] 2) Preparation of enzyme conjugates: using the modified sodium periodate method. 5 mg of HRP was dissolved in 1 ml of 0.2 mol / L, pH 5.6 acetate buffer, 0.1 ml of 1% DNFB in anhydrous ethanol was added, and the mixture was gently stirred at room temperature for 1 h. 0.5 mL of freshly prepared 0.1 mol / L NaIO4 was added, at which point the solution changed from original brown to dark green. The mixture was placed at 4°C for 30 min, at which point the solution changed from original brown to dark green. 1 ml of 2.5% ethylene glycol was added, and the reaction was gently stirred at room temperature for 1 h to terminate the reaction. 10 mg of the humanized Aβ oligomer antibody HAC1-4 prepared in Example 6 was added, and the pH was adjusted to 9.0 with 1.0 mol / L, pH 9.5 CB. The mixture was mixed and placed at 4°C overnight. 0.1 ml of sodium borohydride solution was added, mixed, and placed at 4°C for 3 h. The mixture was dialyzed against 0.01 mol / L, pH 7.4 PBS at 4°C overnight, with the solution changed three times. The precipitate was removed by centrifugation at 3000 r / min for 30 min, and the supernatant was collected. The diluent was diluted 1:2000 to obtain the enzyme conjugate solution.

[0132] 3) Preparation of calibrators / quality control materials:

[0133] 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).

[0134] Calibrator solutions S1 to S5 contain Aβ oligomer antigens at different concentrations diluted in calibrator diluent. The concentrations are 31.25 pg / mL, 62.5 pg / mL, 125 pg / mL, 250 pg / mL, and 500 pg / mL, respectively.

[0135] The quality control solutions QC1 and QC2 are solutions containing different concentrations of Aβ oligomer antigen diluted with calibrator diluent. The concentrations are 62.5 pg / mL and 250 pg / mL, respectively.

[0136] 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.

[0137] 5) Luminescent liquid was purchased from Solebol, catalog number PE0010.

[0138] The above components are assembled into a kit.

[0139] 2. How to use the kit

[0140] The Aβ oligomer detection kit prepared above is used in conjunction with a modern fully automatic chemiluminescence analyzer. The detection procedure is as follows:

[0141] 1) Pipette 50 μl of antibody magnetic beads, apply magnetic field to adsorb the beads, and remove the magnetic bead storage solution.

[0142] 2) Pipette 50 μl of sample, mix with magnetic beads and incubate at 37°C for 15 min.

[0143] 3) Apply a magnetic field to adsorb the magnetic beads, remove the sample solution after the reaction, and wash the magnetic beads once with 200 μl of washing solution.

[0144] 4) Pipette 50 μl of enzyme conjugate, mix with magnetic beads, and incubate at 37°C for 15 min.

[0145] 5) Apply a magnetic field to adsorb the magnetic beads to remove the enzyme after the reaction, and wash the magnetic beads twice with 200 μl of washing solution.

[0146] 6) Pipette 50 μl each of luminescent solution A and B, mix with the magnetic beads, incubate at 37°C for 3 min, and measure the luminescence value.

[0147] 3. Detection of specific recognition of Aβ oligomers

[0148] Aβ polypeptide monomers and Aβ oligomer antigens 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 ;from Figure 5 It can be seen that with the increase of antigen molar concentration, the detection signal of Aβ oligomer antigen increases gradually, but the detection signal of Aβ monomer remains low, indicating that the kit established by the present invention can specifically recognize Aβ oligomers but not monomers.

[0149] 4. Performance indicators of the test kit (blank limit, repeatability, specificity)

[0150] 1) Blank limit

[0151] 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.

[0152] 2) Repeatability

[0153] 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.

[0154] Coefficient of variation (CV) = SD / Mean×100%.

[0155] 3) Specificity

[0156] A 500 pg / ml Aβ1-42 polypeptide 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.

[0157] Cross-reaction rate = M / 500 × 100%.

[0158] The above performance index results are shown in Table 8

[0159] Table 8: Performance indicators of Aβ oligomer detection kit

[0160]

[0161] Example 8: Detection and Verification of Aβ Oligomers in Peripheral Blood and Brain Tissue of AD Model Mice

[0162] 1. Sample Preparation

[0163] Three six-month-old male APP / PS1 AD model mice and three wild-type control mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were selected. After anesthesia, cardiac blood was collected and centrifuged with anticoagulant. The upper plasma layer was collected. Brain tissue was removed and 1 mL of RIPA strong lysis reagent (containing protease and phosphatase inhibitors) was added to the brain tissue. The brain tissue was disrupted using a TissueLyser II tissue grinder at 30 Hz for 8 minutes, and then centrifuged at 14,000 rpm at 4°C for 30 minutes. The supernatant was collected as the brain tissue homogenate.

[0164] 2. Detection

[0165] The detection kit and detection procedure prepared in Example 7 were used to detect Aβ oligomers in the plasma and brain tissue homogenates of AD mice and control mice, respectively. The Aβ oligomer content in the peripheral blood and brain tissue of AD model mice should be significantly higher than that of normal mice. The detection results were consistent with expectations, as shown in Figure 7. Figure 6 , as shown in 7.

[0166] Serum samples were collected from the hospital, including 40 normal controls and 20 samples from patients diagnosed with Alzheimer's disease (AD). The test kit and test procedure prepared in Example 7 were used for testing. The results are shown in Tables 9-11 and Figure 8 :

[0167] Table 9: Aβ oligomer detection results in normal subjects (pg / ml)

[0168]

[0169] Table 10: Aβ oligomer detection results of AD patients (pg / ml)

[0170]

[0171] Table 11: Statistical results of normal subjects and AD samples (pg / ml)

[0172]

[0173] The above results show that there is a significant difference between the normal group and the AD 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 AD screening.

[0174] 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. An antibody that binds to Aβ oligomers, characterized in that The antibody contains: An L chain having CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO.1; the amino acid sequence of CDR2 is shown in SEQ ID NO.2; and the amino acid sequence of CDR3 is shown in SEQ ID NO.3; An H chain having CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO.4; the amino acid sequence of CDR2 is shown in SEQ ID NO.5; and the amino acid sequence of CDR3 is shown in SEQ ID NO.

6.

2. The antibody binding to Aβ oligomers according to claim 1, wherein The preparation method of the Aβ oligomer is as follows: dissolving the Aβ polypeptide with DMSO, diluting with PBS, incubating with constant temperature shaking, centrifuging, collecting the supernatant, and freeze-drying to obtain the Aβ oligomer.

3. The antibody binding to Aβ oligomers according to claim 2, characterized in that The pH value of the PBS is 7.4, and the solution is diluted to 1.5-2.5 mg / mL.

4. The antibody binding to Aβ oligomers according to claim 2, characterized in that The constant temperature shaking incubation temperature is 35-40° C., the time is 44-52 hours, and the shaking frequency is 500-1000 rpm.

5. Use of the antibody binding to Aβ oligomers according to claim 1 in the preparation of a product for AD auxiliary diagnosis.

6. A detection kit, characterized in that The antibody that binds to Aβ oligomers according to claim 1 is used as a raw material.

7. Use of the detection kit according to claim 6 in preparing products for AD auxiliary diagnosis.

Images