Antibody combined with A beta oligomer and application thereof

Abstract

The invention provides an antibody combined with an A beta oligomer and application of the antibody, and belongs to the technical field of medicines. The antibody comprises an L chain with CDR1, CDR2 and CDR3, the amino acid sequence of the L chain of the CDR1 is as shown in SEQ ID NO.1, and the amino acid sequence of the L chain of the CDR2 is as shown in SEQ ID NO.2. The amino acid sequence of the L chain of the CDR2 is as shown in SEQ ID NO. 2; the amino acid sequence of the L chain of the CDR3 is as shown in SEQ ID NO.3; an H chain with CDR1, CDR2 and CDR3 is provided, and the amino acid sequence of the H chain of the CDR1 is as shown in SEQ ID NO.4; and ammonia of the H chain of CDR2. The invention provides a monoclonal antibody capable of being combined with an A beta oligomer as well as a preparation method and application of the monoclonal antibody. The monoclonal antibody is used as a raw material, a unique repeated epitope sandwich method is adopted, and high-specificity, high-sensitivity and high-accuracy detection can be carried out on the A beta oligomer.

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 an application 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, visual-spatial ability impairment, abstract thinking and calculation impairment, personality and behavioral changes, etc. Alzheimer's disease is the most common type of dementia in the elderly, which may account for 60-70% of the cases. And with age, the risk of disease is also increasing. With the acceleration of the global aging process, the number of AD patients is increasing, which has brought a heavy burden to individuals, families and society.

[0003] Amyloidosis occurs in the brain and is the main feature of Alzheimer's disease. Many experts believe that amyloid plaques (Aβ; mainly Aβ1-40 and Aβ oligomers) are closely related to the pathophysiology of AD. According to recent studies, amyloid β oligomers (AβO), soluble aggregates, rather than amyloid fibrils, are more relevant to the pathophysiology of AD.

[0004] Compared with monomers and fibers, AβO is the most neurotoxic, damaging neurons and synapses, leading to memory loss, and is more closely associated with the disease than other markers. Blood AβO levels increase in early subjective cognitive decline (SCD) and mild cognitive impairment (MCI) before the onset of AD. Tracking and detecting AβO at the onset of AD and at different stages during the onset of the disease is of great significance for the early detection, early diagnosis, and disease course monitoring of AD. 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 a raw material and a unique repeated 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 in this way: The present invention provides an antibody that binds to Aβ oligomers, the antibody comprising: 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; 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; the amino acid sequence of the H chain of CDR2 is as shown in SEQ ID NO.5; and the amino acid sequence of the H chain of CDR3 is as shown in SEQ ID NO.6.

[0007] As a further improvement of the present invention, the preparation method of the Aβ oligomer is as follows: Aβ polypeptide is dissolved with DMSO, diluted with PBS, incubated with constant temperature shaking, centrifuged, supernatant is collected, and freeze-dried to obtain Aβ oligomer.

[0008] 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.

[0009] As a further improvement of the present invention, the temperature of the constant temperature oscillation incubation is 35-40°C, the time is 44-52h, and the oscillation frequency is 500-1000rpm.

[0010] The present invention further protects the use of the above-mentioned antibody binding to Aβ oligomers in the preparation of AD auxiliary diagnosis products.

[0011] The present invention further protects a detection kit, which uses the above-mentioned antibody binding to Aβ oligomer as a raw material.

[0012] The present invention further protects the use of the above detection kit in the preparation of AD auxiliary diagnosis products.

[0013] The present invention has the following beneficial effects: 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. The monoclonal antibody is used as a raw material and a unique repeating epitope sandwich method is adopted to detect Aβ oligomers with high specificity, high sensitivity and high accuracy.

[0014] The present invention uses Aβ polypeptide monomers as raw materials and oscillates at a constant temperature of 37°C to aggregate them, uses the prepared Aβ oligomers as immunogens to immunize mice, takes mouse spleen cells to prepare hybridoma cells, screens cells that can secrete and specifically recognize Aβ oligomers by an indirect ELISA method, and prepares and purifies monoclonal antibodies from the cell lines for the detection of Aβ oligomers, which can be used for the auxiliary diagnosis of Alzheimer's disease.

[0015] 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

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative labor.

[0017] Figure 1 This is the electrophoresis diagram of monoclonal antibody AC1-4; Figure 2 It is the titer diagram of anti-Aβ oligomer monoclonal antibody; Figure 3 This is the Western result of monoclonal antibody AC1-4; Figure 4 This is a comparison chart of antibody activity; Figure 5 is the assay specificity map; Figure 6 This is a comparison of plasma Aβ oligomer content between AD and normal mice; Figure 7 This is a comparison of the Aβ oligomer content in brain tissues of AD and normal mice; Figure 8 This is a comparison chart between normal people and AD patients. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Example 1. Preparation of Aβ oligomers 1. 5 mg of Aβ42 peptide (purchased from Hubei Qiangyao Biotechnology) was dissolved in hexafluoroisopropanol (HFIP) to make a 1 mg / ml solution, which was sealed at room temperature for 60 min, placed on ice for 10 min, and then placed in a fume hood with the lid open to evaporate overnight at room temperature to allow the HFIP to evaporate completely.

[0020] 2. Add a small amount of dimethyl sulfoxide (DMSO) to the polypeptide tube after the above treatment to re-dissolve, dilute with PBS to 2 mg / ml, and then place it on a 37 degree constant temperature oscillator and set the oscillation frequency to 800 rpm.

[0021] 3. After shaking for 48 hours, centrifuge at 10,000 rpm for 30 minutes at 4 degrees. The supernatant is Aβ oligomers.

[0022] 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.

[0023] (2) Immunization method: Immunization is performed by subcutaneous multiple-point injection. Freund's complete adjuvant is used 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.

[0024] (3) Immunization dose: 0.1 mg / mouse / time.

[0025] (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 blood was centrifuged at 3000 rpm for 10 min, and the supernatant was collected and packaged and stored at -20°C.

[0026] (5) The Aβ oligomers were coated on an ELISA 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.

[0027] Table 1: Determination of mouse polyclonal antibody titer

[0028] According to the preliminary test results of ELISA, mice numbered 3 had the best immune effect.

[0029] 2. Cell fusion and hybridoma cell screening.

[0030] (1) Recovery and culture of SP2 / 0 tumor cells. Take out the SP2 / 0 tumor cells from the liquid nitrogen tank, dissolve them in a 37°C water bath, transfer them to a centrifuge bottle, add RPMI1640 / 10 preheated at 37°C, centrifuge at 1000rpm for 10min, discard the supernatant, break up the cell clumps, add 10mL RPMI1640 / 10 to the centrifuge bottle, mix well, and place in a 5% CO2, 37°C incubator for culture.

[0031] (2) Preparation of feeder cells. Take an unimmunized Bal b / c mouse, remove its eyeballs and collect blood, then kill the mouse by pulling its neck and soak it in 75% alcohol. Use sterile forceps and scissors to carefully cut the mouse peritoneum to fully expose the abdominal cavity. Use a 10mL sterile syringe to draw 10mL of cold HAT culture medium and inject it into the mouse's abdominal cavity. Use an alcohol cotton ball to gently squeeze the mouse's abdominal cavity to withdraw the culture medium containing feeder cells.

[0032] (3) Preparation of spleen cells. Take the first immunized Balb / c mouse with good serum titer, remove the eyeball to collect positive blood, pull the neck to kill the mouse, and soak it in 75% alcohol. Then, take the spleen of the mouse with the abdomen facing up, grind the spleen with sterile scissors and forceps, then take 5mL of preheated GNK washing solution and slowly add it to the nylon mesh gauze to wash the spleen cells and make a single cell suspension. Then transfer it to a 50mL centrifuge tube, centrifuge at 1000rpm for 10min, discard the supernatant, break up the cell clumps, and resuspend the cells for later use.

[0033] (4) Cell fusion and screening of positive hybridoma cells. Select SP2 / 0 tumor cells in the logarithmic growth phase, transfer them to a 50 mL centrifuge bottle, centrifuge and discard the supernatant, break up the cell clumps, add preheated GNK wash solution, mix the prepared spleen cells and tumor cells in the same centrifuge bottle at a ratio of 6:1-10:1, centrifuge and discard the supernatant, break up the cell clumps. Place in a 37°C water bath, and add 1 mL of preheated polymerizer PEG-1500, shake the centrifuge tube while adding, let stand at 37°C for 5 min, add GNK solution to 40 mL, centrifuge at 1200 rpm for 15 min, discard the supernatant, break up the cell clumps, add 40 mL of preheated HAT selective culture medium, mix the cells evenly, add to the culture plate, 100 µL / well, and culture in a 5% CO2, 37°C incubator.

[0034] (5) When the fused cells in the wells of the culture plate grow to 1 / 3 of the bottom of the well, take the supernatant and screen the positive hybridoma cells by indirect ELISA. Select cells with high titer and good sensitivity and transfer them 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 grow to 1 / 2 of the bottom of the well, take the supernatant to measure the titer value (Table 2).

[0035] Table 2: Titers of the screened monoclonal antibodies

[0036] Five hybridoma cell lines with relatively high titers were screened and obtained, among which AC1-4 had the highest titer.

[0037] (1) Sterilized liquid paraffin was injected intraperitoneally into multiparous female Bal b / c mice, and the five positive hybridoma cells were expanded and cultured respectively. When the cells were in the logarithmic growth phase, the positive hybridoma cells AC1-4 prepared in Example 2 were diluted and injected intraperitoneally. About 7-10 days later, the abdomen of the mouse was significantly enlarged, and there was a sense of tightness when touched by hand, and the ascites was collected.

[0038] (2) The stored 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 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 value 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 precipitate was resuspended in PBS and dialyzed at 4°C for 12 h, during which the solution was changed 2-3 times. The dialysate (purified antibody) was collected and stored at -20°C for future use.

[0039] 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 size of the heavy chain and light chain of IgG, indicating that the method can be used to purify antibodies secreted from AC1-4 hybridoma cells, and was named monoclonal antibody AC1-4. (1) The purified anti-Aβ oligomer monoclonal antibody was tested for its binding to the Aβ oligomer antigen using an enzyme-linked immunosorbent assay to verify the biological activity of the monoclonal antibody. The specific operation is as follows:

[0040] Aβ oligomer antigen was diluted to 1 μg / mL with carbonate buffer (CB, pH 9.6), added to a 96-well microplate at 100 μL / well, and placed at 4°C overnight. The liquid in the wells was discarded, and 200 μL of blocking solution (0.02M PBS, 1% BSA, 5% sucrose) was added to each well and incubated at 37°C for 2 hours. The liquid in the wells was discarded and dried to obtain the antigen-coated plate.

[0041] Sheep anti-mouse secondary antibody labeled with HRP: modified sodium periodate method was used. Take 5 mg of HRP and dissolve it in 0.5 ml of purified water solution, add 0.5 mL of freshly prepared 0.1 mol / L NaIO4, and place it at 4 ° C for 30 minutes. At this time, the solution changes from original brown to dark green; add 0.5 ml of 2.5% ethylene glycol, react at room temperature in the dark for 30 minutes, and terminate the reaction; add 5 mg of the antibody to be labeled, use 1.0 mol / L, pH9.5 carbonate buffer (CB), adjust the pH value to 9.0; mix well, place at 4 ° C overnight; add 0.2 ml of 5 mg / ml sodium borohydride solution, mix well, and place at 4 ° C for 2 hours; dialyze overnight at 4 ° C with 0.01 mol / L, pH7.4 PBS, change the solution 3 times; centrifuge at 8000 rpm for 10 min to remove the precipitate; collect the supernatant to obtain HRP labeled secondary antibody.

[0042] Different concentrations of anti-Aβ oligomer monoclonal antibodies were added to the antigen-coated plate, incubated at 37°C for 1 hour, and washed 3 times. 100 μL of 1:5000 diluted HRP-labeled secondary antibody was added, incubated at 37°C for 1 hour, and washed 3 times. 100 μL of TMB colorimetric solution was added, incubated at 37°C for 15 minutes, 50 μL of stop solution was added, and the absorbance value was measured in an ELISA reader (at 450 nm).

[0043] 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 0.12ng / ml.

[0044] (2) Use western blotting to verify the specificity of anti-Aβ oligomer monoclonal antibodies.

[0045] a) Preparation of electrophoresis gel Prepare 10% separation gel and 5% concentration gel according to Table 3 and Table 4.

[0046] Table 3: 10% separation gel formula

[0047] Table 4: 5% concentrated gel formula

[0048] b) Sample electrophoresis Mix 40μl protein and 10μl 5× loading buffer to make the loading solution. Add protein marker and protein sample in sequence, and add 20ul of sample solution to each well. Fill the electrophoresis buffer, cover the tank cover, turn on the power supply, and use 70V constant voltage electrophoresis for about 30 minutes. When the indicator bromophenol blue enters the separation gel, switch to 90V constant voltage electrophoresis. When the indicator reaches about 0.5cm from the bottom of the gel, turn off the power supply and take out the gel plate.

[0049] C) Transfer Wet the PVDF membrane with anhydrous methanol for more than 30 seconds, then rinse with dd H2O for 2 minutes and soak in transfer buffer for 5 minutes. Assemble the transfer "sandwich" structure in 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), and remove the bubbles between each layer. Then transfer the sandwich to the electrophoresis tank, black side to black side. Connect the positive and negative electrodes, place the transfer box into the electrotransfer instrument in the direction of the membrane toward the positive electrode, add transfer buffer, place the electrotransfer instrument in ice water, and transfer the membrane at 200mA constant current for 90 minutes. After the transfer is completed, quickly remove the PVDF membrane and place it in 5% BSA at room temperature for 2 hours. Wash the membrane with TBST for 5 minutes × 3 times.

[0050] d) Closed Prepare blocking solution: add 2.5g skim milk powder to 50ml freshly prepared TBST solution and shake well. Pour some TBST solution on the cutting plate, place the PVDF membrane on the cutting plate, mark it, and cut the membrane. Add blocking solution and place it on a horizontal shaker. Incubate at room temperature for 60 minutes, remove the blocked membrane, and rinse the hybridization membrane 3 times with washing solution.

[0051] e) Antibody incubation After blocking, add primary antibody to the hybridized membrane and incubate overnight at 4°C. Wash the membrane three times with 1×TBST solution, 10 min each time. Add secondary antibody and incubate at 4°C for 2 h.

[0052] f) Development Remove the membrane from the TBST buffer, remove the excess buffer on the membrane, lay it flat on a piece of cardboard with the protein side facing up, and add the prepared working diluent. Cover the membrane. Incubate the membrane with the working diluent for 1-5 minutes, ensuring that the entire surface is covered. Remove the excess liquid, wrap the PVDF membrane with plastic wrap, and fix it with X-ray film in a dark room.

[0053] like Figure 3 , channel 1 is Aβ oligomer antigen, channel 2 is Aβ monomer antigen, and channel 3 is blood sample of AD patient. Anti-Aβ oligomer monoclonal antibody can recognize both Aβ oligomer antigen and Aβ monomer. Furthermore, the result of channel 3 shows that this antibody can specifically recognize Aβ oligomer in human blood sample.

[0054] 1) The total RNA of hybridoma cells AC1-4 was extracted by Trizol method and cDNA was synthesized using the reverse transcription kit of BD SMARTTM.

[0055] 2) The amplification primer (5'-CTCAGGGAARTARCCYTTGAC-3') was designed based on the mouse antibody heavy chain constant region sequence and the adapter primer in the kit for PCR amplification to obtain the anti-Aβ oligomer mouse monoclonal antibody heavy chain fragment, which was sequenced after the pGEM-T vector was constructed.

[0056] 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 for PCR amplification to obtain the anti-Aβ oligomer mouse monoclonal antibody light chain fragment, which was sequenced after the pGEM-T vector was constructed.

[0057] 4) CDR analysis of anti-Aβ oligomer mouse monoclonal antibody AC1-4.

[0058] According to the sequences of the heavy chain and light chain variable regions of the AC1-4 antibody obtained by sequencing, and referring to the website http: / / www.bioinf.org.uk / abs / #cdrdef for the definition method of antibody CDR analysis, the CDR sequence of the light chain variable region of the anti-oligomer mouse monoclonal antibody AC1-4 was obtained, see Table 5. The CDR sequence of the heavy chain variable region of the anti-Aβ oligomer mouse monoclonal antibody AC1-4 was obtained, see Table 6.

[0059] Table 5: CDR sequences of the light chain variable region of anti-Aβ oligomer mouse monoclonal antibody AC1-4

[0060] Table 6: CDR sequences of the heavy chain variable region of anti-Aβ oligomer mouse monoclonal antibody AC1-4

[0061] 5) Obtain the sequence of humanized monoclonal antibody AC1-4 The obtained framework regions (HFR1, HFR2, HFR3, HFR4) of the heavy chain variable region and the framework regions (LFR1, LFR2, LFR3, LFR4) of the anti-Aβ oligomer mouse monoclonal antibody AC1-4 were compared with the IgBlast search database (IMGT human Vgenes (F+ORF+in-frameP)) to obtain the human FR region with the highest homology, and further consider the domain amino acids of the human framework region 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: The light chain variable region of the humanized anti-Aβ oligomer monoclonal antibody HAC1-4 is shown in SEQ ID NO: 7.

[0062] The heavy chain variable region of the humanized anti-Aβ oligomer monoclonal antibody HAC1-4 is shown in SEQ ID NO: 8.

[0063] 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.

[0064] 1. Preparation of humanized anti-Aβ oligomer mouse monoclonal antibody HAC1-4.

[0065] 1. Construction of eukaryotic expression vector The variable region of humanized anti-Aβ oligomer mouse monoclonal antibody HAC1-4 was connected to the human IgG constant region (Fc) gene to form the full-length heavy and light chain genes, and then recombined into the eukaryotic expression vector pcDNA3.1 through the corresponding restriction sites.

[0066] The details are as follows: The vector for expressing the antibody HAC1-4 light chain is a vector 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; 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); The amino acid sequence of the light chain of the above monoclonal antibody HAC1-4 consists of SEQ ID NO: 7 and SEQ ID NO: 9 (human IgG light chain constant region).

[0067] The vector expressing the heavy chain of antibody HAC1-4 is a vector obtained by replacing the DNA molecule composed of the heavy chain variable region of humanized anti-Aβ oligomer mouse monoclonal antibody HAC1-4 and the heavy chain constant region gene of human IgG with the restriction sites between BamHI and EcoRI of the eukaryotic expression vector pcDNA3.1; the vector expresses the heavy chain of monoclonal antibody HAC1-4; The nucleotide sequence of the DNA molecule consisting of the humanized anti-Aβ oligomer mouse 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); 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).

[0068] 2. Eukaryotic expression of antibodies 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, and then the linearized vector expressing the antibody HAC1-4 light chain and the linearized vector expressing the antibody HAC1-4 heavy chain were electroporated together at 300V and 900μF using a BioRadXcell 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, and the medium was replaced once every 2 days for 2 weeks, and the culture medium was collected.

[0069] The culture fluid was centrifuged to collect the cell culture supernatant, and the antibodies in the supernatant were detected by indirect ELISA method: 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, and incubated at 37°C for 1 hour. After washing, the substrate TMB was added for color development, and sulfuric acid was added to terminate the reaction, and the absorbance was measured by a microplate reader (at 450nm).

[0070] The results are shown in Table 7 below. It can be seen that the cell supernatants with different numbers all contain AC1-4 monoclonal antibody.

[0071] Table 7: Antibody test results

[0072] 3. Purification of humanized anti-Aβ oligomer monoclonal antibodies HAC1-4.

[0073] Affinity purification was performed using Beaver Bio Magrose Protein A Antibody Purification Magnetic Beads.

[0074] (1) Magnetic bead pretreatment: Vortex the antibody purification magnetic beads for 30 seconds to fully resuspend the magnetic beads; take 10% (v / v) magnetic bead suspension and place it in another new 1.5mL 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.

[0075] Note: The amount of magnetic beads in this step can be adjusted according to the maximum binding amount of the magnetic beads to 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 less than 70µg / mL, in order to improve the protein recovery rate, the amount of magnetic beads can be increased, such as increasing it to 3 times the amount of magnetic beads.

[0076] (2) Protein adsorption: Add the magnetic beads pretreated in the above step to 1 ml of sample solution, shake well, and place in a flip mixer to mix at 2-8°C overnight to allow the sample and magnetic beads to fully contact and adsorb. Collect the magnetic beads by magnetic separation and discard the supernatant.

[0077] (3) Magnetic bead washing: Add 1 mL of Binding / Washing buffer to the centrifuge tube, vortex to resuspend the magnetic beads, perform magnetic separation, and discard the supernatant; repeat this operation 3 times.

[0078] (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 an inverting mixer at room temperature (about 25°C) or gently invert the tube manually. Perform magnetic separation after inverting for 10 minutes and collect the supernatant into a new centrifuge tube.

[0079] Note: For the amount of Elution buffer used in this step, it 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 proteins will remain on the magnetic beads during the first elution, resulting in a decrease in protein recovery rate.

[0080] (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 to maintain the biological activity of the protein and avoid protein inactivation.

[0081] (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.

[0082] The purified humanized anti-Aβ oligomer monoclonal antibody HAC1-4 was sequenced, and the HAC1-4 monoclonal antibody was contained, and the sequences of the light chain and heavy chain of the HAC1-4 monoclonal antibody were consistent with the light chain and heavy chain sequences of the aforementioned HAC1-4, proving that the expressed antibody was the HAC1-4 monoclonal antibody.

[0083] 2. Detection of biological activity of humanized and mouse anti-Aβ oligomer monoclonal antibodies HAC1-4 / AC1-4.

[0084] The difference in binding specificity of the purified humanized anti-Aβ oligomer monoclonal antibodies HAC1-4 obtained above to the Aβ oligomer antigen was determined by enzyme-linked immunosorbent assay to verify the biological activity of the humanized monoclonal antibodies. The specific operation is as follows: 1) Dilute the Aβ oligomer antigen to 1 μg / mL with sodium bicarbonate buffer (pH 9.5), coat the ELISA plate, block it, wash it, and dry it for later use; 2) Add 100 μl of the purified humanized anti-Aβ oligomer monoclonal antibody HAC1-4 obtained above (diluted to 2 μg / ml, the dilution buffer is pH 7.4, 0.02 mol / L PBS buffer), 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, add sulfuric acid to terminate the reaction and measure the absorbance using an enzyme reader (at 450 nm).

[0085] 5) Mouse monoclonal antibody AC1-4 and HRP-labeled goat anti-mouse IgG were used as controls to compare the differences in the measurement values ​​of the two antibodies.

[0086] 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 retains the biological activity against Aβ oligomer antigen.

[0087] 1. Preparation of the kit The magnetic microparticle chemiluminescence kit is prepared using the HAC1-4 antibody, and the components include: HAC1-4 antibody magnetic beads, enzyme conjugates, calibrators / quality control products, washing solutions and luminescent solutions.

[0088] 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, activate at room temperature for 30 min, 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 3 times with PBS containing 0.01% Tween 20, and finally added to 1 ml of PBS containing 1% BSA for storage.

[0089] 2) Preparation of enzyme conjugates: using the modified sodium periodate method. Take 5 mg of HRP and dissolve it in 1 ml of 0.2 mol / L, pH 5.6 acetate buffer, add 0.1 ml of 1% DNFB anhydrous ethanol solution, and stir gently at room temperature for 1 hour; add 0.5 mL of freshly prepared 0.1 mol / L NaIO4, at which time the solution changes from original brown to dark green, and place it at 4°C for 30 minutes, at which time the solution changes from original brown to dark green; add 1 ml of 2.5% ethylene glycol, stir gently at room temperature for 1 hour to terminate the reaction; add 10 mg of humanized Aβ oligomer antibody HAC1-4 prepared in Example 6, adjust the pH value to 9.0 with 1.0 mol / L pH 9.5 CB; mix well, and place at 4°C overnight; add 0.1 ml of sodium borohydride solution, mix well, and place at 4°C for 3 hours; dialyze overnight at 4°C with 0.01 mol / L, pH 7.4 PBS, change the solution 3 times; centrifuge at 3000 r / min for 30 min to remove the precipitate; collect the supernatant. The diluent was diluted 1:2000 to obtain the enzyme conjugate solution.

[0090] 3) Preparation of calibrators / quality control products: The calibrator diluent was PBS buffer at pH 7.4, 0.02 M, containing 1% BSA and 0.02% (volume percentage) Proclin-300, which served as the zero calibrator (S0).

[0091] The calibrator solutions S1 to S5 are solutions containing different concentrations of Aβ oligomer antigen diluted with the calibrator diluent. The concentrations are 31.25 pg / mL, 62.5 pg / mL, 125 pg / mL, 250 pg / mL, and 500 pg / mL, respectively.

[0092] The quality control solutions QC1 and QC2 are solutions containing different concentrations of Aβ oligomer antigen diluted with the calibration solution. The concentrations are 62.5 pg / mL and 250 pg / mL respectively.

[0093] 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, 0.5 ml Tween 20, and add purified water to make up to 1 L.

[0094] 5) Luminescent liquid was purchased from Solebol, catalog number PE0010.

[0095] The above components are assembled into a kit.

[0096] 2. How to use the kit The Aβ oligomer detection kit prepared above is used in conjunction with a 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.

[0097] 2) Pipette 50 μl of sample, mix with magnetic beads and incubate at 37 degrees for 15 minutes.

[0098] 3) Add 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.

[0099] 4) Pipette 50 μl of enzyme conjugate, mix with magnetic beads and incubate at 37 degrees for 15 minutes.

[0100] 5) Add a magnetic field to adsorb the magnetic beads, remove the enzyme after the reaction, and wash the magnetic beads twice with 200μl washing solution.

[0101] 6) Pipette 50 μl of luminescent solution A and B respectively, mix with magnetic beads, incubate at 37 degrees for 3 minutes, and detect the luminescence value.

[0102] 3. Detection of specific recognition of Aβ oligomers Aβ polypeptide monomers and Aβ oligomer antigens were diluted with diluent to 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 in 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.

[0103] 4. Performance indicators of the test kit (blank limit, repeatability, specificity) 1) Blank limit Use the kit prepared as above, use the zero-concentration calibrator as the sample for detection, repeat the measurement 20 times, obtain the absorbance values ​​(A values) of the 20 measurement results, calculate the average value (M) and standard deviation (SD), and obtain the A value corresponding to M+2SD; according to the dose-response curve of the calibrator used in the kit, substitute the A value corresponding to M+2SD into the curve, and find the corresponding concentration value, which is the blank limit.

[0104] 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.

[0105] Coefficient of variation (CV) = SD / Mean×100%.

[0106] 3) Specificity A 500 pg / ml Aβ1-42 polypeptide solution was used as a specific sample and tested using the kit prepared above. The test was repeated twice and the average value (M) of the results was calculated.

[0107] Cross-reaction rate = M / 500×100%.

[0108] The above performance index results are shown in Table 8 Table 8: Performance indicators of Aβ oligomer detection kit

[0109] Example 8: Detection and verification of Aβ oligomers in peripheral blood and brain tissue of AD model mice 1. Sample Preparation Three male APP / PS1 AD model mice aged 6 months and three wild-type control mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were selected. After anesthesia, blood was collected from the heart, anticoagulant was added and centrifuged, and the upper plasma was collected. At the same time, brain tissue was removed, 1 mL of RIPA strong lysing agent (containing protease inhibitors and phosphatase inhibitors) was added to the brain tissue, and the brain tissue was crushed with a TissueLyserⅡ tissue grinder at a frequency of 30 Hz for 8 minutes, and centrifuged at 14000 rpm for 30 minutes at 4°C. The supernatant was collected as the brain tissue homogenate.

[0110] 2. Detection 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, such as Figure 6 , as shown in 7.

[0111] Serum samples were collected from the hospital, including 40 normal controls and 20 samples of patients clinically 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 : Table 9: Aβ oligomer detection results in normal people (pg / ml)

[0112] Table 10: Aβ oligomer detection results of AD patients (pg / ml)

[0113] Table 11: Statistical results of normal subjects and AD samples (pg / ml)

[0114] From the above results, it can be seen 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 by this method can be used for AD screening.

[0115] 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 principle of the present invention should be included in the protection scope 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 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; 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; the amino acid sequence of the H chain of CDR2 is as shown in SEQ ID NO.5; and the amino acid sequence of the H chain of CDR3 is as shown in SEQ ID NO.

6.

2. The antibody binding to Aβ oligomer according to claim 1, characterized in that 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 oligomer according to claim 2, characterized in that The pH value of the PBS solution is 7.4, and the dilution is to 1.5-2.5 mg / mL.

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

5. Use of the antibody binding to Aβ oligomers as claimed in claim 1 in the preparation of products for AD auxiliary diagnosis.

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

7. Use of the detection kit as claimed in claim 6 in the preparation of products for AD auxiliary diagnosis.

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