A kit for detecting beta-amyloid by double antibody sandwich and application thereof

Abstract

The application belongs to the field of disease detection, and particularly relates to a double antibody sandwich kit for detecting beta-amyloid and application thereof. The kit uses a capture antibody 5D8, the heavy chain variable region of which comprises HCDR1-3 as shown in SEQ ID NO: 3-5, and the light chain variable region of which comprises LCDR1-3 as shown in SEQ ID NO: 6-8; the kit uses a detection antibody 7H11, the heavy chain variable region of which comprises HCDR1-3 as shown in SEQ ID NO: 11-13, and the light chain variable region of which comprises LCDR1-3 as shown in SEQ ID NO: 14-16. The detection kit has high sensitivity and strong specificity for detecting beta-amyloid, and has important significance for the diagnosis and prevention of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of disease detection, specifically relating to a double-antibody sandwich assay kit for detecting β-amyloid protein and its application. Background Technology

[0002] With the increasing aging of the global population, the incidence of neurodegenerative diseases is rising year by year, among which Alzheimer's disease (AD) has become one of the major diseases seriously affecting the quality of life of the elderly. AD is a chronic, progressive central nervous system disease, whose main clinical manifestations include memory loss, cognitive impairment, and behavioral abnormalities. It is estimated that there are currently about 50 million AD patients worldwide, and this number is expected to continue to grow in the coming decades, placing a heavy burden on society and families.

[0003] In studies of the pathological mechanisms of Alzheimer's disease (AD), abnormal deposition of amyloid-β (Aβ) is considered a key factor in inducing and promoting disease progression. Aβ is a neurotoxic polypeptide fragment produced by the enzymatic hydrolysis of amyloid precursor protein (APP). Under normal physiological conditions, the production and clearance of Aβ maintain a dynamic balance. However, in the brains of AD patients, increased Aβ production and decreased clearance lead to its abnormal deposition in brain tissue, forming senile plaques, which in turn trigger a series of pathological processes such as neuroinflammation, oxidative stress, and neuronal damage.

[0004] Due to the crucial role of β-amyloid protein in the pathogenesis of Alzheimer's disease (AD), its detection technology is of great significance in clinical diagnosis, disease monitoring, and evaluation of treatment efficacy. Currently, although various β-amyloid protein detection methods are applied in research and clinical practice, they still have limitations in terms of ease of operation, detection speed, cost-effectiveness, and sensitivity. Therefore, researching a novel and efficient β-amyloid protein detection method is of great practical significance and application value for improving the diagnostic accuracy of AD and achieving early intervention and treatment. This invention is based on this background, aiming to provide a highly sensitive and specific β-amyloid protein detection technology to contribute to the prevention and treatment of AD. Summary of the Invention

[0005] This invention provides a double-antibody sandwich assay kit for detecting human β-amyloid protein. The capture antibody used in the kit is 5D8, whose heavy chain variable region includes HCDR1-3 as shown in SEQ ID NO: 3-5, and whose light chain variable region includes LCDR1-3 as shown in SEQ ID NO: 6-8. The detection antibody used in the kit is 7H11, whose heavy chain variable region includes HCDR1-3 as shown in SEQ ID NO: 11-13, and whose light chain variable region includes LCDR1-3 as shown in SEQ ID NO: 14-16.

[0006] Preferably, the heavy chain variable region of the 5D8 antibody is as shown in SEQ ID NO: 1.

[0007] Preferably, the light chain variable region of the 5D8 antibody is as shown in SEQ ID NO: 2.

[0008] Preferably, the heavy chain variable region of the 7H11 antibody is as shown in SEQ ID NO: 9.

[0009] Preferably, the light chain variable region of the 7H11 antibody is as shown in SEQ ID NO: 10.

[0010] Preferably, the kit further includes an enzyme-labeled plate, a blocking solution, a washing solution, and a colorimetric solution.

[0011] Preferably, the detection antibody is labeled with horseradish peroxidase.

[0012] In a preferred embodiment of the present invention, a double-antibody sandwich detection method for detecting human β-amyloid protein is provided, comprising the following steps:

[0013] 1. Coating: Dilute 5D8 with 1×PBS to 2μg / mL, mix well using a vortex mixer, and incubate at 4℃ overnight at 100μL / well.

[0014] 2. Blocking: Wash the plate three times with PBST, and block with 3% skim milk powder, 300 μL / well, at 37℃ for 1 h;

[0015] 3. Add sample: Discard the blocking solution, wash the plate three times with PBST, add 1 μg of Aβ(1-42) polymer or β-amyloid fibrils, use PBS buffer as a negative control, 100 μL / well, incubate at 37℃ for 1 h;

[0016] 4. Add detection antibody: Dilute HRP-labeled 7H11 to 0.5 μg / mL, add 100 μL / well to each well of the microplate, cover with the cover film, and incubate at 37°C for 1 h.

[0017] 5. Color development: Wash the plate six times with PBST, add 100 μL of color development solution to each well, and develop the color at 37℃ for 15 min;

[0018] 6. Terminate the reaction: Add 50 μL of 2M H2SO4 to each well to terminate the reaction, and read the value at OD450nm using a microplate reader.

[0019] Preferably, the 5D8 heavy chain variable region includes HCDR1-3 as shown in SEQ ID NO: 3-5, and the light chain variable region includes LCDR1-3 as shown in SEQ ID NO: 6-8.

[0020] Preferably, the 7H11 heavy chain variable region includes HCDR1-3 as shown in SEQ ID NO: 11-13, and the light chain variable region includes LCDR1-3 as shown in SEQ ID NO: 14-16.

[0021] The detection kit constructed in this invention has high sensitivity and strong specificity in detecting β-amyloid protein, which is of great significance for the diagnosis and prevention of Alzheimer's disease.

[0022] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0023] Figure 1 This is a scatter plot of β-amyloid protein detected by a double-antibody sandwich ELISA.

[0024] Figure 2 This is a graph showing the specific detection results of β-amyloid protein by a double-antibody sandwich ELISA. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0026] Example 1: Preparation of immune complexes

[0027] 1. Experimental materials

[0028] Human Aβ synthesized via solid-phase method (1-42) Its amino acid sequence is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA, purchase β-amyloid fibrils;

[0029] 1M IPTG: Weigh 1.2g of IPTG into a beaker, add 40mL of ddH2O and stir thoroughly to dissolve it. Add an appropriate amount of ddH2O to make up to 50mL. Filter using a 0.22μm filter and dispense into sterile 1.5mL centrifuge tubes. Store at -20℃ for later use.

[0030] Binding Buffer: Mix 20 mL of 1 M Tris-HCl, 0.34 g imidazole, 29.22 g NaCl and 480 g urea in a beaker, add an appropriate amount of deionized water and stir thoroughly to dissolve completely. Adjust the pH to 8.0 and then add an appropriate amount of deionized water to bring the volume to 1 L.

[0031] Elution Buffer: Mix 20 mL of 1 M Tris-HCl, 34 g of imidazole, 29.22 g of NaCl and 480 g of urea in a beaker, add an appropriate amount of deionized water and stir thoroughly to dissolve completely. Adjust the pH to 8.0 and then add an appropriate amount of deionized water to bring the volume to 1 L.

[0032] Membrane transfer buffer: Mix 5.8g Tris-base, 0.37g SDS, 2.9g Glycine and 200mL methanol in a beaker, add an appropriate amount of deionized water, stir to dissolve and bring the volume to 1L;

[0033] Blocking solution: Add 1.5g BSA to 50mL TBST buffer and stir to dissolve.

[0034] 2. Aβ (1-42) Preparation of polymers

[0035] Aβ (1-42) First, dissolve it in NaOH (0.5 mM) and sonicate for 30 seconds, then add a certain amount of monomer Aβ. (1-42) Dilute in PBS (50 mM NaH2PO4, 100 mM NaCl, pH 7.6) and incubate at 37°C for 6 hours to prepare Aβ. (1-42) Polymer.

[0036] 3. Aβ (1-42) Preparation of binding antigens of polymers and BSA

[0037] Aβ was oxidized with glutaraldehyde. (1-42) Multimer coupled to carrier protein BSA

[0038] Take 2mg Aβ (1-42) The polymer was dissolved in 50 mM NaH2PO4 and allowed to stand for 30 min to dissolve completely.

[0039] According to BSA: Aβ (1-42)Add BSA at a weight ratio of 2:1 (30 mg / ml) to the polymer, then slowly add 100 μl of glutaraldehyde solution while blowing the mixture to ensure uniform mixing. Crosslink the mixture at room temperature for 3 hours, then dissolve it in PBS buffer and store it at -20°C.

[0040] Example 2 Aβ (1-42) Preparation of monoclonal antibodies

[0041] SPF-grade 6-8 week old female Balb / c mice were selected and Aβ was used. (1-42) The multimer and BSA-binding antigen was administered via subcutaneous injection at multiple sites on the back of each mouse at 50 μg / 0.3 mL. Before the first immunization, the immunogen was mixed with an equal volume of complete Freund's adjuvant (purchased from Sigma) to prepare an emulsion. 21 days later, the emulsified antigen was administered via subcutaneous and intraperitoneal injection at 50 μg / 0.5 mL per mouse. 10 days after the second immunization, blood was collected and serum was separated. The titer of the mice was determined by ELISA. Mice with high titers were selected and intraperitoneally injected again with 100 μg / 0.5 mL per mouse of the antigen. 3 days later, spleen cells were collected for fusion.

[0042] Spleen cell isolation: Under aseptic conditions in a biosafety cabinet, remove a culture dish, add 30-40 mL of basal culture medium, place a cell sieve inside, remove the spleen and place it in the sieve. Remove excess connective tissue and fat from the spleen tissue, mince the spleen tissue, and grind it in the cell sieve. Use a clean grinding stick to press and grind the tissue. The intracellular cells will slowly disperse and, after passing through the cell sieve, will be suspended in the culture dish solution. Wash the cell sieve with 10 mL of basal culture medium and collect the basal culture medium outside the cell sieve. Centrifuge at 400g for 5 min at room temperature, discard the supernatant, retain the cells, add 13 mL of room temperature RBC lysis buffer, gently disperse the cell clumps with a pipette and time for 1 min to lyse the red blood cells, add 37 mL of basal culture medium, mix well, stop red blood cell lysis, centrifuge at 400g for 5 min at room temperature, discard the supernatant, retain the cells, add 40 mL of room temperature basal culture medium, gently disperse the cell clumps with a pipette to resuspend the cells, complete the first wash, centrifuge at 400g for 5 min at room temperature, discard the supernatant, retain the cells, add 20 mL of room temperature basal culture medium, gently disperse the cell clumps with a pipette to resuspend the cells; filter the resuspended cells again through a cell sieve to remove clumped cells, and then count the cells;

[0043] Tissue suspension was filtered through a copper mesh to obtain single spleen cells. Freshly extracted spleen cells and SP2 / 0 cells cultured to the logarithmic growth phase were counted; 2.0–5.0 × 10⁶ cells were collected from each cell. 7After mixing SP2 / 0 with spleen cells, centrifuge and discard the supernatant. Slowly and evenly add PEG, repeating this process 5 times with DMEM. Centrifuge again on the last addition and discard the supernatant. Carefully resuspend the cell pellet in HAT selection medium and seed it into 96-well tissue culture plates containing feeder cells. Add 0.1 ml (based on spleen cells, the volume per well is 2 × 10⁶ cells) to each well. 5 (Number of cells). One well was filled with SP2 / 0 containing HAT-selective culture medium as a control well for SP2 / 0's sensitivity to HAT. The cells were incubated at 37°C in a 5% CO2 incubator. Around day seven after cell confluence, all cells in the SP2 / 0 control well died. Large hybridoma cell clones were visible after 10-14 days. Once the culture medium turned slightly yellow, a portion of the supernatant was aspirated for analysis.

[0044] Further testing was conducted on the affinity of the monoclonal antibodies in the 42 positive clones, and the two strains with the best affinity were selected and named 5D8 and 7H11.

[0045] Table 2 Affinity constants of monoclonal antibodies

[0046] 5D8 <![CDATA[1.35×10 -4 ]]> <![CDATA[3.58×10 5 ]]> <![CDATA[3.77×10 -10 ]]> 7H11 <![CDATA[3.79×10 -4 ]]> <![CDATA[9.54×10 5 ]]> <![CDATA[3.97×10 -10 ]]>

[0047] Sequencing was performed on 5D8 and 7H11. The heavy chain variable region sequence of 5D8 is as follows:

[0048] QVELQQPGAELVRPQSSVKLSCKASQYTFT SYGMH WVKQRPIQGLEWIG SIKNNGSTTTYAPSLKG KATLTVDKSSSTAYMQLSSLTSEDSAVYYCAR AVSGLGIVYYPYYMA WGQGTSVTVSS(SEQ ID NO: 1);

[0049] The light chain variable region sequence of 5D8 is as follows

[0050] DIQMTQTTSSLSASLGDRVTISC RLVHSNTRCG WYQQKPDGTIKLLIY YDCDRFS GVPSRFSGSGSGTDYSLTISNLEPEDIATYYC WCYHHVP FGTGTKLELK(SEQ ID NO: 2);

[0051] The underlined characters indicate the CDRs of 5D8, where VH CDR1 is SYGMH (SEQ ID NO: 3), VH CDR2 is SIKNNGSTTTYAPSLKG (SEQ ID NO: 4), VH CDR3 is AVSGLGIVYYPYYMA (SEQ ID NO: 5), VL CDR1 is RLVHSNTRCG (SEQ ID NO: 6), VL CDR2 is YDCDRFS (SEQ ID NO: 7), and VL CDR3 is WCYHHVP (SEQ ID NO: 8).

[0052] The heavy chain variable region sequence of 7H11 is

[0053] QVQLQQPGAELVKPGASVKLSCKASGYTFT LSYCA WVKQRPGQGLEWIG NSRPDSLPAAID KATLTVDKSSSTAYMQLSSLTSEDSAVYYCAR TGKAEV WGQGTTLTVSS(SEQ ID NO: 9);

[0054] The light chain variable region sequence of 7H11 is as follows:

[0055] DIVLTQSPASLAVSLGQRAIISC RAGDG WYQQKPGQQPKLLIY YDCDGSNGDVK GVPTRFSGSGSRTDFTLNIHPVEEDDAATYYC YQSIHHPWP FGGGTKLEIK (SEQ ID NO: 10);

[0056] The underlined characters indicate the CDRs of 7H11, where VH CDR1 is LSYCA (SEQ ID NO: 11), VH CDR2 is NSRPDSLPAAID (SEQ ID NO: 12), VH CDR3 is TGKAEV (SEQ ID NO: 13), VL CDR1 is RAGDG (SEQ ID NO: 14), VL CDR2 is YDCDGSNGDVK (SEQ ID NO: 15), and VL CDR3 is YQSIHHPWP (SEQ ID NO: 16).

[0057] Example 3: Detection of β-amyloid protein by double-antibody sandwich ELISA

[0058] Aβ detection method based on double-antibody sandwich enzyme-linked immunosorbent assay using anti-mouse monoclonal antibodies 5D8 and 7H11 (1-42) The method for producing polymeric or β-amyloid fibrils specifically includes the following steps:

[0059] 1. Coating: Dilute 5D8 with 1×PBS to 2μg / mL, mix well using a vortex mixer, and incubate at 4℃ overnight at 100μL / well.

[0060] 2. Blocking: Wash the plate three times with PBST, and block with 3% skim milk powder, 300 μL / well, at 37℃ for 1 h;

[0061] 3. Add sample: Discard the blocking solution, wash the plate three times with PBST, and add 1 μg Aβ. (1-42) Multimers or β-amyloid fibrils were incubated at 37°C for 1 hour with PBS buffer as a negative control at 100 μL / well.

[0062] 4. Add detection antibody: Dilute HRP-labeled 7H11 to 0.5 μg / mL, add 100 μL / well to each well of the microplate, cover with the cover film, and incubate at 37°C for 1 h.

[0063] 5. Color development: Wash the plate six times with PBST, add 100 μL of color development solution to each well, and develop the color at 37℃ for 15 min;

[0064] 6. Terminate the reaction: Add 50 μL of 2M H2SO4 to each well to terminate the reaction, and read the value at OD450nm using a microplate reader.

[0065] like Figure 1 As shown, this application uses Aβ (1-42) Monoclonal antibodies 5D8 and 7H11, prepared from multimers as immunogens, bind to Aβ. (1-42) The fibrils have strong ability to detect Aβ using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) established with 5D8 and 7H11. (1-42) The detection limit for fibrils was 15.6 pg / ml.

[0066] To investigate the effect of the aforementioned double-antibody sandwich enzyme-linked immunosorbent assay method on Aβ (1-42) The specificity of fibrils, with 1 ml of Aβ (1-42) Multimer, Aβ (1-40) Monomer, Tau protein, BSA, and PBS buffer were used as antigen samples for double antibody sandwich assay.

[0067] The results are as follows Figure 2 As shown, the double-antibody sandwich detection method established by 5D8 and 7H11 of the present invention exhibits good specificity for Aβ(1-42) fibrils.

[0068] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A double-antibody sandwich assay kit for detecting human β-amyloid protein, wherein the capture antibody used in the kit is 5D8, the heavy chain variable region of which includes HCDR1-3 as shown in SEQ ID NO: 3-5, and the light chain variable region of which includes LCDR1-3 as shown in SEQ ID NO: 6-8; and the detection antibody used in the kit is 7H11, the heavy chain variable region of which includes HCDR1-3 as shown in SEQ ID NO: 11-13, and the light chain variable region of which includes LCDR1-3 as shown in SEQ ID NO: 14-16.

2. The double antibody sandwich assay kit according to claim 1, characterized in that, The heavy chain variable region of the 5D8 antibody is shown in SEQ ID NO:

1.

3. The double antibody sandwich assay kit according to claim 1, characterized in that, The light chain variable region of the 5D8 antibody is shown in SEQ ID NO:

2.

4. The double antibody sandwich assay kit of claim 1, wherein, The heavy chain variable region of the 7H11 antibody is shown in SEQ ID NO:

9.

5. The double antibody sandwich assay kit of claim 1, wherein, The light chain variable region of the 7H11 antibody is shown in SEQ ID NO:

10.

6. The double antibody sandwich assay kit of claim 1, wherein, The kit also includes an enzyme-labeled plate, blocking solution, washing solution, and colorimetric solution.

7. The double antibody sandwich assay kit of claim 1, wherein, The detection antibody was labeled with horseradish peroxidase.

8. An antibody combination for detecting human β-amyloid, characterized in that, The antibody combination consists of capture antibody 5D8 and detection antibody 7H11. The 5D8 heavy chain variable region includes HCDR1-3 as shown in SEQ ID NO: 3-5, and the light chain variable region includes LCDR1-3 as shown in SEQ ID NO: 6-8. The 7H11 heavy chain variable region includes HCDR1-3 as shown in SEQ ID NO: 11-13, and the light chain variable region includes LCDR1-3 as shown in SEQ ID NO: 14-16.

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