Kit for double-antibody sandwich detection of beta-amyloid protein and application of kit

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of disease detection, and particularly relates to a kit for detecting β-amyloid protein by double antibody sandwich and its application. Background Art

[0002] With the aggravation of the aging of the global population, the incidence of neurodegenerative diseases has been increasing year by year. Among them, Alzheimer's Disease (AD) has become one of the main diseases seriously affecting the quality of life of the elderly. AD is a chronic and progressive central nervous system disease, and its main clinical manifestations include memory loss, cognitive dysfunction, behavioral abnormalities and other symptoms. According to statistics, there are currently about 50 million AD patients globally, and this figure is expected to continue to grow in the next few decades, bringing a heavy burden to society and families.

[0003] In the research of the pathological mechanism of AD, the abnormal deposition of β-amyloid protein (Aβ) is considered to be a key factor inducing and promoting the development of the disease. 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, the production of Aβ increases and the clearance decreases, resulting in its abnormal deposition in brain tissues, forming senile plaques, and then triggering a series of pathological processes such as neuroinflammation, oxidative stress, and neuronal damage.

[0004] Due to the important role of β-amyloid protein in the pathogenesis of AD, its detection technology is of great significance in clinical diagnosis, disease monitoring and evaluation of treatment effects. At present, although there are already a variety of β-amyloid protein detection methods applied in scientific research and clinical practice, they still have certain limitations in terms of operational simplicity, detection speed, cost-effectiveness, sensitivity, etc. Therefore, researching a new and efficient method for detecting β-amyloid protein has great practical significance and application value for improving the diagnostic accuracy of AD and realizing early intervention and treatment. Based on this background, the present invention is committed to providing a β-amyloid protein detection technology with high sensitivity and good specificity, in order to contribute to the prevention and control and treatment of AD. Summary of the Invention

[0005] The present invention provides a double-antibody sandwich detection kit for detecting human β-amyloid protein. The capture antibody used in the kit is 5D8, and its heavy-chain variable region contains HCDR1-3 as shown in SEQ ID NO: 3-5, and its light-chain variable region contains LCDR1-3 as shown in SEQ ID NO: 6-8; the detection antibody used in the kit is 7H11, and its heavy-chain variable region contains HCDR1-3 as shown in SEQ ID NO: 11-13, and its light-chain variable region contains 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-linked immunosorbent assay (ELISA) plate, a blocking solution, a washing solution, and a chromogenic solution.

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

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

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

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

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

[0016] 4. Adding detection antibody: Dilute HRP-labeled 7H11 to 0.5 μg / mL, then add 100 μL per well to the ELISA plate in sequence, cover with a 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 developer to each well and incubate at 37 °C for 15 min;

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

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

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

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

[0022] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the drawings to fully understand the purpose, features and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 2 is a specific detection result graph of detecting β-amyloid protein by double antibody sandwich ELISA. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The present invention will be described in detail below in conjunction with the drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0026] Example 1 Preparation of Immune Complex

[0027] 1. Experimental materials

[0028] Synthesize human Aβ by solid-phase method (1-42) , whose amino acid sequence is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA, and purchase β-amyloid fibrils;

[0029] 1M IPTG: Weigh 1.2 g of IPTG and place it in a beaker. Add 40 mL of ddH₂O and stir well to dissolve it. Add an appropriate amount of ddH₂O to make the volume up to 50 mL. Filter it through a 0.22-μm filter and dispense it into sterilized 1.5-mL centrifuge tubes, and store it at -20 °C for later use.

[0030] Binding Buffer: Take 20 mL of 1M Tris-HCl, 0.34 g of imidazole, 29.22 g of NaCl and 480 g of urea, mix them in a beaker, add an appropriate amount of deionized water and stir well until completely dissolved. After adjusting the pH to 8.0, add an appropriate amount of deionized water to make the volume up to 1 L.

[0031] Elution Buffer: Take 20 mL of 1M Tris-HCl, 34 g of imidazole, 29.22 g of NaCl and 480 g of urea, mix them in a beaker, add an appropriate amount of deionized water and stir well until completely dissolved. After adjusting the pH to 8.0, add an appropriate amount of deionized water to make the volume up to 1 L.

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

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

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

[0035] Dissolve Aβ (1-42) first in NaOH (0.5 mM) and sonicate for 30 seconds. Then dilute a certain amount of monomeric Aβ (1-42) in PBS (50 mM NaH₂PO₄, 100 mM NaCl, pH 7.6) and incubate at 37 °C for 6 hours to prepare Aβ (1-42) multimers.

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

[0037] Couple Aβ (1-42) multimers to the carrier protein BSA using glutaraldehyde

[0038] Take 2 mg of Aβ (1-42) multimers and dissolve it in 50 mM NaH₂PO₄, let it stand for 30 min to completely dissolve it;

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

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

[0041] Select SPF-grade female Balb / c mice at 6 - 8 weeks old, and use the binding antigen of Aβ (1-42) polymer and BSA for subcutaneous injection at multiple points on the back at 50 μg / 0.3 mL / mouse. Before the first immunization, mix the immunogen with an equal amount of complete Freund's adjuvant (purchased from Sigma) to make an emulsifier; 21 days later, after emulsifying the antigen, perform subcutaneous and intraperitoneal injections at 50 μg / 0.5 mL / mouse; 10 days after the second immunization, collect blood to separate serum, and measure the titer of mice by ELISA method. Select mice with high titers and inject 100 μg / 0.5 mL / mouse antigen intraperitoneally again; 3 days later, take spleen cells for fusion;

[0042] Isolate spleen cells: Under sterile operation in a safety cabinet, take out a culture dish, add 30 - 40 mL of basic medium, place a cell sieve, take out the spleen and place it in the cell sieve. Cut off the excess connective tissue and fat on the spleen tissue, cut the spleen tissue into pieces and grind it in the cell sieve. Take a clean grinding rod and roll and grind the tissue with the end of the pressing part. The cells in the membrane will slowly free out. After passing through the cell sieve, they will be suspended in the culture dish solution; wash the cell sieve with 10 mL of basic medium and collect the basic medium outside the cell sieve. Centrifuge at a centrifugal force of 400 g for 5 min at room temperature, discard the supernatant, keep the cells, add 13 mL of normal-temperature RBC lysate, gently disperse the cell mass with a pipette and start timing for 1 min for RBC lysis. Add 37 mL of basic medium, mix well to terminate RBC lysis. Centrifuge at a centrifugal force of 400 g for 5 min at room temperature, discard the supernatant, keep the cells, add 40 mL of normal-temperature basic medium, gently disperse the cell mass with a pipette to resuspend the cells, complete the first wash. Centrifuge at a centrifugal force of 400 g for 5 min at room temperature, discard the supernatant, keep the cells, add 20 mL of normal-temperature basic medium, gently disperse the cell mass with a pipette to resuspend the cells; filter the resuspended cells through the cell sieve again to remove clumped cells, and then count the cells;

[0043] Filter the tissue suspension with a copper mesh to obtain single spleen cells, and count the freshly extracted spleen cells and SP2 / 0 cells cultured to the logarithmic phase; respectively take 2.0 - 5.0×10 7After mixing SP2 / 0 with splenocytes, centrifuge and discard the supernatant. Slowly and evenly add PEG, repeat adding DMEM 5 times, and centrifuge for the last time to remove the supernatant. Carefully resuspend the cell pellet with HAT selection medium and inoculate it into the wells of a 96-well tissue culture plate containing feeder cells. Add 0.1 ml to each well (calculated based on splenocytes, the amount added to each well is 2×10 5 cells). Leave one well to add SP2 / 0 containing HAT selection medium as a sensitivity control well for SP2 / 0 to HAT. Incubate in an incubator at 37°C with 5% CO2. Around the seventh day after cell fusion, all the cells in the SP2 / 0 control well die. Large hybridoma cell clones can be seen after 10 - 14 days. When the culture medium turns slightly yellow, part of the supernatant can be aspirated for detection.

[0044] Further detect the affinity of monoclonal antibodies in the above 42 positive clones, and screen out the 2 with the best affinity, named 5D8 and 7H11.

[0045] Table 2 Affinity constants of monoclonal antibodies

[0046] Monoclonal antibody Koff (l / s) Kon (l / Ms) KD (M) 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] Sequence 5D8 and 7H11. The variable region sequence of the heavy chain of 5D8 is

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

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

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

[0051] The CDRs of 5D8 are underlined, 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

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

[0056] The CDRs of 7H11 are underlined, 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 Double Antibody Sandwich ELISA for Detecting β - Amyloid Protein

[0058] A double antibody sandwich enzyme - linked immunosorbent assay for detecting Aβ based on anti - mouse monoclonal antibodies 5D8 and 7H11 (1-42) Method for detecting multimers or β - amyloid fibrils, specifically including the following steps:

[0059] 1. Coating: Dilute 5D8 with 1×PBS to 2 μg / mL. After mixing well with a vortex mixer, add 100 μL per well and incubate overnight at 4°C.

[0060] 2. Blocking: Wash the plate three times with PBST, then block with 3% skim milk, adding 300 μL per well and incubating at 37°C for 1 h.

[0061] 3. Adding samples: Discard the blocking solution, wash the plate three times with PBST, then add 1 μg Aβ (1-42) multimers or β-amyloid fibrils, using PBS buffer as a negative control, adding 100 μL per well and incubating at 37°C for 1 h.

[0062] 4. Adding detection antibodies: Dilute HRP-labeled 7H11 to 0.5 μg / mL, then add 100 μL per well to the enzyme-linked immunosorbent assay (ELISA) plate in sequence. Cover with a cover film and incubate at 37°C for 1 h.

[0063] 5. Color development: Wash the plate six times with PBST, then add 100 μL of chromogenic solution per well and develop color at 37°C for 15 min.

[0064] 6. Terminating the reaction: Add 50 μL of 2 M H2SO4 to each well to terminate the reaction, and read the absorbance value at OD450nm with an ELISA reader.

[0065] As Figure 1 shown, the monoclonal antibodies 5D8 and 7H11 prepared using Aβ (1-42) multimers as immunogens have strong ability to bind Aβ (1-42) fibrils. The detection limit of the double-antibody sandwich enzyme-linked immunosorbent assay for Aβ (1-42) fibrils established with 5D8 and 7H11 is 15.6 pg / ml.

[0066] To explore the specificity of the double-antibody sandwich enzyme-linked immunosorbent assay for Aβ (1-42) fibrils, use 1 ml of Aβ (1-42) multimers, Aβ (1-40) monomers, Tau protein, BSA, and PBS buffer as antigen samples for double-antibody sandwich detection.

[0067] Results Figure 2 as shown indicate that the double-antibody sandwich detection method established with 5D8 and 7H11 of the present invention exhibits good specificity for Aβ(1-42) fibrils.

[0068] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field based on the concept of the present invention through logical analysis, reasoning, or limited experiments on the basis of the prior art shall fall within the protection scope determined by the claims.

Claims

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

2. The double-antibody sandwich detection kit according to claim 1, wherein The heavy chain variable region of the 5D8 antibody is as shown in SEQ ID NO:

1.

3. The double-antibody sandwich detection kit according to claim 1, wherein The light chain variable region of the 5D8 antibody is as shown in SEQ ID NO:

2.

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

9.

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

10.

6. The double-antibody sandwich detection kit according to claim 1, wherein The kit further includes an enzyme-linked immunosorbent assay (ELISA) plate, a blocking solution, a washing solution, and a chromogenic solution.

7. The double-antibody sandwich detection kit according to claim 1, wherein, The detection antibody is labeled with horseradish peroxidase (HRP).

8. A double-antibody sandwich detection method for detecting human β-amyloid protein, comprising the following steps: (1) Coating: Dilute 5D8 with 1×PBS to 2 μg / mL, mix well with a vortex mixer, add 100 μL per well, and incubate overnight at 4°C. (2) Blocking: Wash the plate three times with PBST, block with 3% skim milk powder, add 300 μL per well, and incubate at 37°C for 1 h. (3) Adding sample: Discard the blocking solution, wash the plate three times with PBST, add 1 μg Aβ(1-42) polymer or β-amyloid fibril, use PBS buffer as a negative control, add 100 μL per well, and incubate at 37°C for 1 h. (4) Adding detection antibody: Dilute HRP-labeled 7H11 to 0.5 μg / mL, then add 100 μL per well to the ELISA plate in sequence, cover with a cover film, and incubate at 37°C for 1 h. (5) Chromogenic reaction: Wash the plate six times with PBST, add 100 μL of chromogenic solution to each well, and develop color at 37°C for 15 min. (6) Terminating the reaction: Add 50 μL of 2M H2SO4 to each well to terminate the reaction, and read the value at OD450nm with an ELISA reader.

9. The double-antibody sandwich detection method according to claim 8, wherein, The 5D8 heavy chain variable region contains HCDR1-3 shown in SEQ ID NO: 3-5, and the light chain variable region contains LCDR1-3 shown in SEQ ID NO: 6-8.

10. The double-antibody sandwich detection method according to claim 8, characterized in that, The 7H11 heavy chain variable region contains HCDR1-3 shown in SEQ ID NO: 11-13, and the light chain variable region contains LCDR1-3 shown in SEQ ID NO: 14-16.

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