Preparation of A beta 1-40 or A beta 1-42 recombinant antibody and application of A beta 1-40 or A beta 1-42 recombinant antibody in Alzheimer's disease joint detection kit

Abstract

The invention provides preparation of an A [beta] 1-40 or A [beta] 1-42 recombinant antibody and application of the A [beta] 1-40 or A [beta] 1-42 recombinant antibody in an Alzheimer's disease joint detection kit, and belongs to the technical field of Alzheimer's disease detection, and the preparation method comprises the following steps: screening out a specific antibody aiming at A [beta] 1-40 and A [beta] 1-42 proteins, and successfully expressing the recombinant anti-A [beta] 1-40 and A [beta] 1-42 antibody with high affinity by using an HEK293 cell. According to the prepared A beta 1-40 and A beta 1-42 joint detection kit, a flow fluorescence technology is utilized, a joint detection method for detecting the levels of A beta 1-40 and A beta 1-42 of serum is established through a double-antibody sandwich method, and the kit has important application value and significance for large-scale early screening of AD.

Description

Technical Field

[0001] The present invention relates to the technical field of Alzheimer's disease detection, and in particular to the preparation of recombinant antibodies against Aβ1-40 or Aβ1-42 and their application in a combined Alzheimer's disease detection kit. Background Art

[0002] Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Once onset, it is irreversible. Early diagnosis, prevention, detection, and treatment are crucial for preventing and delaying the progression of AD. The difficulty in treating AD lies in its unclear pathogenesis. Currently, the widely accepted mechanism is that an imbalance in the production and clearance of amyloid-β (Aβ) is the initiating factor in neuronal degeneration and dementia. Abnormal levels of Aβ form plaques between neurons in the brain, which are neurotoxic and lead to neuronal degeneration. Aβ plaque accumulation is a hallmark pathophysiological characteristic of AD, and the Aβ42 / 40 ratio is used clinically as a reference indicator.

[0003] Aβ 1-40 and Aβ 1-42 are Aβ fragments of different lengths, among which Aβ 1-42 It is more prone to aggregation and plaque formation and has stronger neurotoxicity. Studies have shown that Aβ in the cerebrospinal fluid of AD patients 1-42 Reduced Aβ 1-42 There is a negative correlation between the level of AD and the amount of amyloid plaques in the brain. The accuracy of AD diagnosis has increased to 80% through the detection of biomarkers in cerebrospinal fluid. However, for early screening and widespread population-wide coverage of AD, the use of neuroimaging and cerebrospinal fluid biomarker testing is not practical. Therefore, the development of AD biomarkers based on blood tests is very necessary.

[0004] The patent with announcement number CN114920851B, entitled "Aβ1-42 antigen and its use in detecting Aβ1-42 concentration in Alzheimer's patients," discloses that the Aβ1-42 antigen prepared from a human Aβ1-42 antigen determinant polypeptide can be used to prepare antibodies and detection kits; the amino acid sequence of the human Aβ1-42 antigen determinant polypeptide of this invention is Tyr-Arg-Asp-Gly-Asp-Gly-Asp-Met-Val-Gly-Gly-Val-Val-Ile-Ala. Aβ1-42 antigen can be prepared by coupling human Aβ1-42 antigen determinant polypeptide with protein carrier; Aβ1-42 monoclonal antibody or polyclonal antibody can be prepared from the Aβ1-42 antigen of this invention; Aβ1-42 monoclonal antibody or polyclonal antibody is used to prepare Aβ1-42 in vitro diagnostic kit; the human Aβ1-42 antigen determinant polypeptide of this invention has good antigenicity, and the antigen (immunogen) prepared with it can produce highly specific monoclonal antibodies and polyclonal antibodies in animals, which can be used for in vitro detection of human Aβ1-42. However, the existing technology for Aβ1-42 is not very effective. 1-42 , Aβ 1-40 The preparation of recombinant antibodies and their application in joint detection need further development. Summary of the Invention

[0005] In view of this, the present invention provides a preparation of recombinant Aβ1-40 or Aβ1-42 antibodies and their use in an Alzheimer's disease joint detection kit, so as to achieve the purpose of rapid antibody production, strong stability and convenient large-scale screening of AD.

[0006] To achieve the above objectives, the present invention provides a method for preparing a recombinant antibody against Aβ1-40 or Aβ1-42, comprising the following steps: S1. Aβ1-40 or Aβ1-42 polypeptide is synthesized, mice are immunized with the synthesized Aβ1-40 or Aβ1-42 polypeptide, spleen lymphocytes are isolated from the spleen tissue obtained from the immunized mice, RNA is extracted from the spleen lymphocytes, and cDNA is synthesized after reverse transcription. The cDNA is used as a template, and VH upstream primer, VH downstream primer, VL upstream primer, VL downstream primer are added respectively to perform PCR amplification. The target fragment obtained after amplification is recovered by running on a gel to obtain the VH and VL genes of the Aβ1-40 or Aβ1-42 antibody, and sequenced and identified to obtain the VH and VL sequences and CRD partition sequences of the Aβ1-40-1, Aβ1-40-2 or Aβ1-42 antibody; the VH region sequence of the Aβ1-40-1 is as shown in SEQ ID No. 1, the VL region sequence of the Aβ1-40-1 is as shown in SEQ ID No. 2, and the VH region sequence of the Aβ1-40-2 is as shown in SEQ ID No.3, the VL region sequence of the Aβ1-40-1 is SEQ ID No.4, the VH region sequence of the Aβ1-42 is SEQ ID No.4, and the VL region sequence of the Aβ1-42 is SEQ ID No.5; S2. The obtained VH and VL gene fragments are subjected to a recombination reaction to obtain a VH-linker-VL recombinant plasmid, which is co-cultured with competent cells to extract the plasmid, and the gene is transfected into HEK293 cells to express the antibody. The antibody is separated and purified to obtain Aβ1-40 or Aβ1-42 recombinant antibody.

[0007] Optionally, when immunizing the mice, Aβ1-40 or Aβ1-42 polypeptide is mixed with squalene respectively, and then the mice are immunized by subcutaneous injection, and a second immunization is performed using the same method two weeks later.

[0008] Optionally, the RNA is extracted from the spleen lymphocytes by mixing the spleen lymphocytes with a separation reagent to obtain a mixed solution at a ratio of 2.5×10 7 1 mL of isolation reagent was added to each cell, and 1 mL of the mixture was taken for RNA extraction. The isolation reagent was Tipure Isolation Reagent.

[0009] Optionally, the VH upstream primer is CAGTGTGGTGGAATT-GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG; the VH downstream primer is GCTCAGGGAARTAGCCCTTGAC--ggcggcggcggcagcggcggcggcggcagc; the VL upstream primer is ggcggcggcggcagcggcggcggcggcagc-GAYATTGTGMTSACMCARWCTMCA; the VL downstream primer is TGGGAAGATGGATACAGTT-AATTCCACCACACTG; the PCR amplification conditions are pre-denaturation at 95°C for 3 minutes, followed by 35 cycles of keeping at 95°C for 30 seconds → keeping at 55°C for 30 seconds → keeping at 72°C for 1 minute, and finally keeping at 72°C for 7 minutes.

[0010] Optionally, the VH and VL gene fragments are subjected to a recombination reaction by using EcoRI and HandIII to double-digest a mammalian cell expression vector, and then the VH and VL gene fragments obtained respectively are subjected to a recombination reaction using a seamless ligation kit to obtain a VH-linker-VL recombinant plasmid.

[0011] Alternatively, the plasmid can be extracted by co-culturing with competent cells. Thaw TOP10 competent cells on ice, take 5-10 µL of the cooled recombinant plasmid, add it to 100 µL of competent cells, mix well, place on ice for 15 minutes, heat shock at 42°C for 45-90 seconds, and quickly cool on ice for 2-5 minutes. Add approximately 500 µL of antibiotic-free LB medium, incubate at 37°C, shaking at 200 rpm for 30 minutes, and evenly spread the shaken culture liquid onto an LB-AG solid culture plate. Incubate the plate upside down in a 37°C incubator overnight. Extract the plasmid using a plasmid extraction kit.

[0012] Optionally, the gene is transfected into HEK293 cells to express the antibody as a plasmid and HEK293 cells are shaken in serum-free medium, and the supernatant is collected after centrifugation to express the expressed antibody.

[0013] In order to achieve the above-mentioned object, the present invention also provides an Alzheimer's disease joint detection kit, comprising a fluorescently labeled antibody solution, wherein the fluorescently labeled antibody solution comprises Aβ1-40 or Aβ1-42 recombinant antibodies obtained by the preparation method of Aβ1-40 or Aβ1-42 recombinant antibodies.

[0014] Optionally, the fluorescently labeled antibody solution includes the following preparation method: S1. Magnetic carboxyl fluorescent encoded microspheres were washed with PBS buffer, EDC and NHS were added and the mixture was shaken in the dark, Aβ1-40-2 recombinant antibody or Aβ1-42 recombinant antibody was added and rotated at room temperature, the fluorescent microspheres were washed with microsphere cleaning solution to remove excess antibody, and skim milk powder was added for blocking. After removing the skim milk powder, Tris buffer was added for storage to prepare the antibody solution coupled to the microspheres; S2. Take the Aβ1-40 recombinant antibody 1 and phycoerythrin fluorescein, shake and incubate at room temperature, and wash with PBS solution to remove excess unbound phycoerythrin fluorescein to obtain a fluorescently labeled antibody solution.

[0015] Optionally, the microsphere washing solution includes PBS buffer, pH 7.4, 0.1% BSA, 0.02% Tween 20, and 0.05% Proclin 300.

[0016] The above technical solution of the present invention includes at least the following beneficial effects: 1. The technical solution provided by the present invention utilizes genomic recombinant antibodies to increase the production speed of antibodies with higher affinity and stability.

[0017] 2. The present invention prepares a combined detection kit for Aβ1-40 and Aβ1-42, and uses flow cytometry technology to establish a combined detection method for detecting serum Aβ1-40 and Aβ1-42 levels through a double antibody sandwich method, which has important application value and significance for large-scale early screening of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the titer of mouse immune serum in Example 1 of the present invention; Figure 2 The Aβ1-40 and Aβ1-42 antibodies obtained in Example 1 of the present invention; Figure 3 This is a graph showing the activity of the Aβ1-40 and Aβ1-42 recombinant antibodies in Example 2 of the present invention; Figure 4 This is a purity chart of the Aβ1-40 and Aβ1-42 recombinant antibodies in Example 2 of the present invention; Figure 5 This is a scatter plot of the concentrations of Aβ1-40 and Aβ1-42 recombinant antibodies in each sample in Example 3 of the present invention. DETAILED DESCRIPTION

[0019] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the following will be combined with the appended drawings of the embodiments of the present invention. Figure 1-5, clearly and completely describing the technical solutions of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the described embodiments of the present invention, all other embodiments derived by ordinary technicians in this field fall within the scope of protection of the present invention.

[0020] Example 1 Synthesis of Aβ1-40 or Aβ1-42 peptides: The Aβ1-40 immunogen is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMV GGVV, Aβ1-42 immunogen is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGVVIA, are all peptides. The peptides used in this study were synthesized by Sangon Biotechnology Co., Ltd.

[0021] Mouse immunization: Aβ1-40 and Aβ1-42 peptides were mixed with the novel immune adjuvant squalene and immunized subcutaneously in 6-8 week-old BALB / c mice at a dose of 50 μg per mouse. The mice were divided into five groups: Mouse 1, Mouse 2, Mouse 3, Mouse 4, and a control group. The control group mice did not receive a subcutaneous injection. Two weeks later, a second immunization was performed using the same method and dose. After the second immunization, tail blood was collected and serum titers were determined by serial dilution ELISA. The results determined whether a booster immunization was necessary.

[0022] One week after the final immunization, mouse blood was collected for serum antibody titer testing. A 96-well microtiter plate was coated with 10 μg / ml Aβ1-40 or Aβ1-42 peptide at 4°C for 24 hours. The plates were then blocked with 1% BSA at 37°C for 1 hour. The plates were then diluted with serially diluted samples and incubated at 37°C for 1 hour. HRP-labeled secondary antibodies were then added and incubated at 37°C for 1 hour. TMB substrate solution was then added and the plates reacted in the dark at room temperature for 15 minutes. The reaction was terminated with 2M sulfuric acid and the OD value at 450 nm was read. The highest dilution with a P / N ratio ≥ 2.1 was used as the serum titer.

[0023] Lymphocyte isolation: Soak the separated spleen tissue in a clean PBS solution and cut it into small pieces with scissors. Add an appropriate amount of PBS solution to a culture dish, place a 200-mesh cell sieve, remove the spleen, and place it in the cell sieve. Take a clean syringe and gently crush the tissue with the end of the syringe. The cells inside the membrane will slowly release and, after passing through the cell sieve, be suspended in the culture dish solution. Rinse the cell sieve with a small amount of PBS and collect the PBS outside the cell sieve. Centrifuge at 400g for 15 minutes. Remove the supernatant, add 3-5ml of red blood cell lysis buffer, resuspend, and incubate at room temperature for 2 minutes. Centrifuge at 400g for 15 minutes. Wash twice with 5ml of PBS and remove the supernatant to obtain splenic lymphocytes.

[0024] RNA extraction and reverse transcription synthesis of cDNA: Add 1 mL of Tipure Isolation Reagent per 2.5 × 107 cells. 1 mL was used for RNA extraction, and the remainder was stored at -80°C. Total RNA was extracted according to the RNA extraction kit's protocol, and the obtained total RNA was used as a template for reverse transcription and cDNA synthesis according to the reverse transcription kit's instructions.

[0025] Construction of mouse immune VH-VL gene library: Using cDNA as template, VH upstream primer {[PCDNA3.1(+) homology arm (15 bp)]CAGTGTGGTGGAATT-GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG}, downstream primer {GCTCAGGGAARTAGCCCTTGAC--ggcggcggcggcagcggcgg cggcggcagc) and VL upstream primer (ggcggcggcggcagcggcggcggcggcagc-GAYATTGTG MTSACMCARWCTMCA), downstream primer {TGGGAAGATGGATACAGTT-AATTCC PCR amplification was performed using the following conditions: initial denaturation at 95°C for 3 minutes, followed by 35 cycles of 95°C for 30 seconds, then 55°C for 30 seconds, then 72°C for 1 minute, followed by a final hold at 72°C for 7 minutes. The target fragment was run on agarose gel, recovered, and sequenced.

[0026] The serum titer during the mouse immunization process of Example 1 was detected. Figure 1 . And for the VH and VL genes of Aβ1-40 or Aβ1-42 antibodies obtained in Example 1, see Figure 2The gene fragments were sequenced and identified as shown in Table 1.

[0027] Depend on Figure 1 It can be seen that Figure 1 A shows that after immunization, all four mice developed corresponding immune titers to Aβ1-40 peptide or Aβ1-42 peptide. For Aβ1-40 peptide, the immune titer P / N value (OD value of immunized mice / OD value of non-immunized mice) of mouse 1 was the highest at the same dilution ( Figure 1 B); For Aβ1-42 peptide, the immune titer P / N value (OD value of immune mouse / OD value of non-immunized mouse) of mouse 3 was the highest at the same dilution ( Figure 1 B). Therefore, mouse 1 (Aβ1-40) and mouse 3 (Aβ1-42) were selected as the subjects for the next experiment.

[0028] Depend on Figure 2 It can be seen that the Aβ1-40 and Aβ1-42 antibody VH and VL genes (500 bp) were successfully retrieved, respectively.

[0029] Sequencing by a genetics company yielded the VH and VL sequences of the Aβ1-40 and Aβ1-42 antibodies. Genentech alignment confirmed that both light and heavy chain variable regions belonged to mouse-derived antibodies. IMGT analysis revealed the corresponding CDR regions (Table 1).

[0030] Table 1 Sequence information of Aβ1-40 and Aβ1-42 antibodies

[0031] Example 2 Construction of VH-linker-VL recombinant plasmid: After double digestion of PCDNA3.1(+) with EcoRI and HandIII, the VH and VL gene segments of the Aβ1-40 or Aβ1-42 recombinant antibodies obtained in Example 1 were subjected to a 15-minute recombination reaction using a seamless ligation kit to obtain a VH-linker-VL recombinant plasmid. Thaw the cloning TOP10 competent cells on ice, take 5-10 μL of the cooled recombinant product, add it to 100 μL of competent cells, mix well, and place on ice for 15 minutes. Heat shock at 42°C for 45-90 seconds, and quickly ice bath for 2-5 minutes. Add approximately 500 μL of antibiotic-free LB medium and culture at 37°C with shaking at 200 rpm for 30 minutes. Take an appropriate volume of bacterial solution and evenly spread it on an LB-AG solid culture plate. Place the plate upside down in a 37°C incubator and culture overnight.

[0032] Antibody expression: Select well-growing positive clones and inoculate them into 5 ml of LB-AG medium at a 1:100 ratio. Incubate overnight at 37°C, 200 rpm. Extract the plasmid using a plasmid extraction kit and transfect the gene into HEK293 cells. Incubate the cells in serum-free medium on a shaker for 5 days. Centrifuge at 10,000 rpm, 4°C for 10 minutes, and collect the supernatant (cell fermentation broth) for antibody purification.

[0033] Antibody purification: After washing the protein G packing material with equilibration buffer (20 mM PBS, 100 mM NaCl, pH 7.4) for approximately 2-3 column volumes, the sample was loaded onto the column at a flow rate of 0.5 mL / min. After loading, the column was washed with wash buffer (20 mM PBS, 100 mM NaCl, pH 7.4) until no protein was detected by Coomassie Brilliant Blue. The target protein was eluted with elution buffer (100 mM glycine, pH 2.5) and adjusted to pH 7.4 with 1 M Tris to obtain Aβ1-40 or Aβ1-42 recombinant antibodies.

[0034] The antibody obtained in Example 2 was tested for activity using the following method: The plates were coated with Aβ1-40 or Aβ1-42 peptides at 1 ug / mL, 150 μl per well, and incubated at 37°C for 1.5 h. The plates were washed 5 times and blocked with 1% BSA, 150 μl per well, and incubated at 37°C for 2 h. The plates were washed 5 times and recombinant Aβ1-40 or Aβ1-42 antibodies diluted at a specific ratio were added, 100 μl per well, and incubated at 37°C for 1.5 h. The plates were washed 5 times and HRP secondary antibody was added, 100 μl per well, and incubated at 37°C for 1.5 h. TMB colorimetric solution was added, 100 μl per well, and color development was performed for about 15 min. The reaction was terminated with stop solution, 50 μl per well. The OD was read at 450 nm and the results were analyzed. Figure 3 . Figure 3 The X-axis shows the antibody reaction concentration from 0 to 1.28 μg / ml, and the Y-axis shows the absorbance (OD450 mm) of Aβ1-40 or Aβ1-42 at 1 μg / ml.

[0035] Depend on Figure 3 It can be seen that the Aβ1-40 or Aβ1-42 recombinant antibodies obtained in the present invention have strong activities, and the activities of the Aβ1-42 recombinant antibody and the Aβ1-40-2 recombinant antibody are stronger than those of the Aβ1-40-1 recombinant antibody.

[0036] The purity of the antibody obtained in Example 2 was identified by preparing a 5% separation gel and a 15% concentration gel, loading the purified antibody onto each sample and performing electrophoresis under constant voltage conditions. When the bromophenol blue reached 1 cm from the bottom edge of the separation gel, the electrophoresis was stopped. After removing the film, place it in Coomassie Brilliant Blue staining solution for 30 min, and finally decolorize it 2 to 3 times with a decolorizing solution until the background is colorless. Scan the film with a scanner and save the image, see Figure 4 , Gelpro32 software was used to analyze the antibody purity.

[0037] Depend on Figure 4 Both Aβ1-40 and Aβ1-42 recombinant antibodies displayed distinct specific bands at 25 kD and 60 kD, with no other contaminants. The total light and heavy chain content (antibody purity) for Aβ1-40 recombinant antibody 1 was 95.18%, for Aβ1-40 recombinant antibody 2 it was 94.62%, and for Aβ1-42 it was 96.46%, demonstrating high antibody purity.

[0038] Determination of the affinity constant of the antibody obtained in Example 2. Method: This study used biofilm interferometry (BLI) to measure the affinity and kinetic parameters of antibody-antigen binding. Antibodies were immobilized on the sensor surface using a Protein G sensor and then reacted with a gradient of antigen dilution. Changes in surface optical interference were analyzed to obtain information about molecular interactions. Specific operating procedures are described in the instrument manual. Results are shown in Table 2.

[0039] Table 2 Affinity test of recombinant antibodies to Aβ1-40 and Aβ1-42

[0040] As shown in Table 2, the affinity of Aβ1-40 recombinant antibody 1 to Aβ1-40 antigen is 3.46×10 -10 M; The affinity of Aβ1-40 recombinant antibody 2 to Aβ1-40 antigen is 7.01×10 -10 The affinity of Aβ1-42 recombinant antibody to Aβ1-42 antigen was 7.01×10 -10 , indicating that the antibody prepared in this experiment has a high affinity to the antigen.

[0041] Identification of the recognition site of the antibody obtained in Example 2: Biopanning of the phage library: Coat one well of a 96-well plate with 150 μL of 100 μg / L avastin overnight at 4°C. After blocking, add 10 μL of the original peptide library diluted in 100 μL of TBST and incubate at room temperature for 1 hour. Rinse 10 times with 0.1% TBST. Add 100 μL of eluent (0.2 M Glycine HCl, 1 g / L LBSA) and incubate for 8 minutes. Aspirate the eluate and neutralize it with 15 μL of 1 M Tris-HCl. Titer 1 μL of the eluate was titered, and the remaining solution was added to 20 mL of Luria-Bertan (LB) medium (containing 200 μL of ER2738 and 20 μL of tetracycline stock solution) for amplification and purification. The second and third rounds of screening were performed according to the above steps, with washes in 0.5% TBST.

[0042] After the third round of screening, 30 well-fragmented phage clones were selected for amplification and purification. The purified phage were added to 96-well plates pre-coated with Avastin and recombinant antibodies against Aβ1-40 or Aβ1-42 for 1 hour at room temperature. HRP-labeled anti-M13 antibody (1:2000) was then added for 1 hour. Color was developed with o-phenylenediamine (OPD), and the A490 nm value was measured. A value 5-fold higher than the negative control was identified as a positive clone.

[0043] 500 μL of the above phage stock solution was added to 200 μL of PEG8000 / NaCl and allowed to stand for 10 minutes. The mixture was centrifuged for 10 minutes, and the supernatant was discarded. The pellet was resuspended in 100 μL of iodide buffer and 250 μL of ethanol was added. The mixture was incubated at room temperature for 10 minutes, centrifuged for 10 minutes, and the supernatant was discarded. The pellet was washed with 70% ethanol and briefly dried under vacuum. The pellet was resuspended in 30 μL of trishydroxymethylaminomethane (Tris) + ethylenediaminetetraacetic acid (EDTA) buffer (10 mM Tris-HCl, 1 mM EDTA). 5 μL of this solution was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

[0044] The results showed that the antigen epitope targeted by Aβ1-40 recombinant antibody 1 was located at DAEFR; the antigen epitope targeted by Aβ1-40 recombinant antibody 2 was located at MVGGV; and the antigen epitope targeted by Aβ1-42 recombinant antibody was located at GVVIA.

[0045] Example 3 The concentration was 5×107 0.1 ml of magnetic carboxyl fluorescent encoded microspheres (Sigma) were washed twice with PBST buffer, 100 μg of EDC and 50 μg of NHS were added and shaken in the dark for 20 minutes, 100 μg of Aβ1-40 recombinant antibody 2 or Aβ1-42 recombinant antibody were added and rotated at room temperature for 2 hours, the fluorescent microspheres were washed with microsphere washing solution (PBS, pH 7.4, 0.1% BSA, 0.02% Tween 20, 0.05% Proclin 300) to remove excess antibodies, and then blocked with 5% skim milk powder for 30 minutes. After removing the skim milk powder, Tris buffer with pH 7.2 was added for storage to prepare the antibody solution coupled to the microspheres.

[0046] Phycoerythrin-conjugated fluorescein Aβ1-40 recombinant antibody 1 and phycoerythrin fluorescein (Sigma) were incubated at a molar ratio of 1:30 with shaking at room temperature for 5 hours. The solution was washed three times with a 50 kD ultrafiltration tube supplemented with 0.01 mol / L PBS solution to remove excess unbound phycoerythrin fluorescein to obtain a fluorescently labeled antibody solution.

[0047] The antibody solution in the prior art kit is replaced with the above-mentioned fluorescent-labeled antibody solution to obtain an Aβ1-40 and Aβ1-42 combined detection kit, and the calibrators are Aβ1-40 and Aβ1-42 antigens (synthetic polypeptides).

[0048] The Aβ1-40 and Aβ1-42 combined detection kit provided in Example 3 was tested: Clinical samples with a fixed gradient were tested to compare the correlation between the test results of the kit and the clinical fixed values. Clinical gradient samples were fixed using Simoa single molecule immunoassay.

[0049] The detection method of the kit is as follows: 25u1 of the test sample, 25u1 of a 100-fold diluted coupled microsphere antibody solution, and 25ul of a 200-fold diluted fluorescent-labeled antibody solution are sequentially added to the flow tube, incubated at 37°C for 2.5 hours, and then washed three times with 200uI of cleaning solution. The fluorescence signal intensity is detected by flow cytometry, and the concentrations of Aβ1-40 and Aβ1-42 in the test sample are calculated based on the Aβ1-40 and Aβ1-42 calibrator curves, respectively.

[0050] The concentration of Aβ1-40 or Aβ1-42 detected by Simoa single molecule immunoassay was used as the horizontal axis, and the fluorescence signal intensity of Aβ1-40 or Aβ1-42 detected by flow cytometry was used as the vertical axis. Scatter plots of the Aβ1-40 and Aβ1-42 concentrations of each sample were drawn, and the correlation coefficients of the Aβ1-40 and Aβ1-42 concentration values ​​between the two detection methods were calculated. Figure 5 .

[0051] like Figure 5 As shown in the figure, the correlation coefficients of the two detection methods are R2=0.9895 (Aβ1-40) and R2=0.9903 (Aβ1-42), respectively. This indicates that when the prepared Aβ1-40 and Aβ1-42 recombinant antibodies are used in flow cytometry experiments with paired antibodies, the detection results have a strong correlation with the Simoa single molecule immunoassay and can be used for downstream kit preparation.

[0052] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a recombinant antibody against Aβ1-40 or Aβ1-42, characterized in that: The following steps are involved: S1. Aβ1-40 or Aβ1-42 polypeptide is synthesized, mice are immunized with the synthesized Aβ1-40 or Aβ1-42 polypeptide, spleen lymphocytes are isolated from the spleen tissue obtained from the immunized mice, RNA is extracted from the spleen lymphocytes, and cDNA is synthesized after reverse transcription. The cDNA is used as a template, and VH upstream primer, VH downstream primer, VL upstream primer, VL downstream primer are added respectively to perform PCR amplification. The target fragment obtained after amplification is recovered by running on a gel to obtain the VH and VL genes of the Aβ1-40 or Aβ1-42 antibody, and sequenced and identified to obtain the VH and VL sequences and CRD partition sequences of the Aβ1-40-1, Aβ1-40-2 or Aβ1-42 antibody; the VH region sequence of the Aβ1-40-1 is as shown in SEQ ID No. 1, the VL region sequence of the Aβ1-40-1 is as shown in SEQ ID No. 2, and the VH region sequence of the Aβ1-40-2 is as shown in SEQ ID No.3, the VL region sequence of the Aβ1-40-1 is SEQ ID No.4, the VH region sequence of the Aβ1-42 is SEQ ID No.4, and the VL region sequence of the Aβ1-42 is SEQ ID No.5; S2. The obtained VH and VL gene fragments are subjected to a recombination reaction to obtain a VH-linker-VL recombinant plasmid, which is co-cultured with competent cells to extract the plasmid, and the gene is transfected into HEK293 cells to express the antibody. The antibody is separated and purified to obtain Aβ1-40 or Aβ1-42 recombinant antibody.

2. The method for preparing the recombinant antibody against Aβ1-40 or Aβ1-42 according to claim 1, characterized in that: When immunizing the mice, Aβ1-40 or Aβ1-42 polypeptides were mixed with squalene respectively, and then the mice were immunized by subcutaneous injection. A second immunization was performed in the same manner two weeks later.

3. The method for preparing a recombinant Aβ1-40 or Aβ1-42 antibody according to claim 1, wherein: The RNA was extracted from the spleen lymphocytes by mixing the spleen lymphocytes with a separation reagent to obtain a mixed solution at a ratio of 2.5×10 7 1 mL of isolation reagent was added to each cell, and 1 mL of the mixture was taken for RNA extraction. The isolation reagent was Tipure Isolation Reagent.

4. The method for preparing the recombinant antibody against Aβ1-40 or Aβ1-42 according to claim 1, characterized in that: The VH upstream primer is CAGTGTGGTGGAATT-GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG; the VH downstream primer is GCTCAGGGAARTAGCCCTTGAC--ggcggcggcggcagcggcggcggcggcagc; the VL upstream primer is ggcggcggcggcagcggcggcggcggcagc-GAYATTGTGMTSACMCARWCTMCA; the VL downstream primer is TGGGAAGATGGATACAGTT-AATTCCACCACACTG; the PCR amplification conditions are pre-denaturation at 95°C for 3 minutes, followed by 35 cycles of keeping at 95°C for 30 seconds → keeping at 55°C for 30 seconds → keeping at 72°C for 1 minute, and finally keeping at 72°C for 7 minutes.

5. The method for preparing the recombinant antibody against Aβ1-40 or Aβ1-42 according to claim 1, characterized in that: The VH and VL gene fragments are subjected to a recombination reaction in which a mammalian cell expression vector is double-digested with EcoRI and HandIII, and then the obtained VH and VL gene fragments are subjected to a recombination reaction using a seamless ligation kit to obtain a VH-linker-VL recombinant plasmid.

6. The method for preparing a recombinant Aβ1-40 or Aβ1-42 antibody according to claim 1, wherein: To extract the plasmid from the competent cells, thaw TOP10 competent cells on ice, take 5-10 µL of the cooled recombinant plasmid, add it to 100 µL of competent cells, mix well, and place on ice for 15 minutes. Then, heat shock at 42°C for 45-90 seconds, and quickly cool on ice for 2-5 minutes. Add approximately 500 µL of antibiotic-free LB medium and incubate at 37°C, shaking at 200 rpm for 30 minutes. Spread the shaken culture liquid evenly on an LB-AG solid culture plate, incubate the plate upside down in a 37°C incubator overnight, and extract the plasmid using a plasmid extraction kit.

7. The method for preparing recombinant Aβ1-40 or Aβ1-42 antibodies according to claim 1, characterized in that: The gene is transfected into HEK293 cells to express the antibody, and the plasmid and HEK293 cells are shaken in serum-free medium for expression, and the supernatant is collected after centrifugation to express the antibody.

8. An Alzheimer's disease joint detection kit, characterized in that: The invention comprises a fluorescently labeled antibody solution, wherein the fluorescently labeled antibody solution comprises the Aβ1-40 or Aβ1-42 recombinant antibody obtained by the preparation method of the Aβ1-40 or Aβ1-42 recombinant antibody according to any one of claims 1 to 7.

9. The Alzheimer's disease joint detection kit according to claim 8, characterized in that: The fluorescent-labeled antibody solution includes the following preparation method: S1. Magnetic carboxyl fluorescent encoded microspheres were washed with PBS buffer, EDC and NHS were added and the mixture was shaken in the dark, Aβ1-40-2 recombinant antibody or Aβ1-42 recombinant antibody was added and rotated at room temperature, the fluorescent microspheres were washed with microsphere cleaning solution to remove excess antibody, and skim milk powder was added for blocking. After removing the skim milk powder, Tris buffer was added for storage to prepare the antibody solution coupled to the microspheres; S2. Take the Aβ1-40 recombinant antibody 1 and phycoerythrin fluorescein, shake and incubate at room temperature, and wash with PBS solution to remove excess unbound phycoerythrin fluorescein to obtain a fluorescently labeled antibody solution.

10. The Alzheimer's disease joint detection kit according to claim 9, characterized in that: The microsphere washing solution includes PBS buffer, pH 7.4, 0.1% BSA, 0.02% Tween 20, and 0.05% Proclin 300.

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