Alzheimer's disease protein marker, antibody diagnostic kit and application of Alzheimer's disease protein marker and antibody diagnostic kit

Abstract

The invention belongs to the technical field of biological medicine, particularly relates to an Alzheimer's disease protein marker, an antibody diagnostic kit and application of the antibody diagnostic kit, and provides the Alzheimer's disease protein marker A beta (1-42). The invention provides a kit for detecting human amyloid A beta based on sandwich ELISA (enzyme-linked immunosorbent assay) of 2B6 and 3A8 antibodies, and further provides a kit for detecting human amyloid A beta based on 2B6 and 3A8 antibodies by predicting B cell epitopes of a template A beta (1-42) protein through ABCpred to reconstruct an immune antigen enhanced Alzheimer disease combined antigen such as SEQ ID NO: 1, called AD-A beta 2 for short, and monoclonal antibodies 2B6 and 3A8 with strong affinity and high titer based on the antigen. The A beta concentration detection line effect of at least 0.5 ng / mL is realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an Alzheimer's disease protein marker and antibody diagnostic kit and applications thereof. Background Art

[0002] Alzheimer's Disease (AD) is a common neurodegenerative disease. With the acceleration of the global aging process, its incidence continues to rise, and it has become a major challenge to public health. The early stages of AD usually manifest as mild cognitive impairment, but the lack of clear biological diagnostic criteria makes it difficult for patients to receive effective intervention in the reversible stage of the disease. Accurate detection of markers is an important tool for early diagnosis and prognosis of the disease, and it is of great significance in screening, efficacy evaluation and disease classification. Therefore, exploring specific markers for Alzheimer's disease has become a current research hotspot.

[0003] Monoclonal antibodies are widely used in the detection of protein markers such as specific markers of Alzheimer's disease due to their strong specificity, high affinity and good batch stability. Current detection technologies mainly rely on methods such as ELISA, Western Blot or immunofluorescence, among which monoclonal antibodies are the core tools in the detection process. However, due to the cross-interference of specific markers of Alzheimer's disease in various forms (such as monomers, oligomers and fibrils), detection methods that rely solely on a single antibody may be insufficient in specificity and sensitivity.

[0004] Some antibodies may cross-react with their precursor proteins when detecting markers, resulting in false positive results. Especially in complex body fluid samples (such as serum or cerebrospinal fluid), the impact of nonspecific binding signals on detection accuracy is more significant. Although monoclonal antibodies have become an important tool for marker detection, their limitations in specificity and sensitivity still need to be improved through antibody optimization, multi-antibody combined detection, or combination with new detection platforms (such as digital PCR or mass spectrometry). Summary of the invention

[0005] In order to solve the above problems, the present invention first provides an Alzheimer's disease protein marker, which is the Aβ(1-42) protein as shown in SEQ ID NO: 1. This marker can provide a clear target for the study of Alzheimer's disease and lay the foundation for the subsequent antigen peptide design, antibody preparation and detection method development.

[0006] In certain embodiments, the present invention further provides an antigenic peptide, which is composed of the antigenic epitope peptide of the Alzheimer's disease protein marker Aβ(1-42) shown in SEQ ID NO: 2-4. By selecting the key epitope peptide segment of Aβ(1-42), an immune response can be induced more effectively for the preparation of specific antibodies and related research.

[0007] In certain embodiments, the amino acid sequence of the antigenic peptide is shown in SEQ ID NO: 5. By optimizing the sequence of the antigenic peptide, its immunogenicity and specificity can be further improved, and the targeting effect of subsequent antibodies can be enhanced.

[0008] The present invention also provides a monoclonal antibody against Aβ(1-42) protein, which has significant specificity and high affinity.

[0009] In certain embodiments, the present invention provides a monoclonal antibody against Aβ(1-42), a protein marker for Alzheimer's disease, wherein the monoclonal antibody is 2B6. The heavy chain variable region of monoclonal antibody 2B6 comprises H-CDR1 as shown in SEQ ID NO:7, H-CDR2 as shown in SEQ ID NO:8, and H-CDR3 as shown in SEQ ID NO:9, and the light chain variable region comprises L-CDR1 as shown in SEQ ID NO:11, L-CDR2 as shown in SEQ ID NO:12, and L-CDR3 as shown in SEQ ID NO:13.

[0010] In certain embodiments, the monoclonal antibody 2B6 consists of a heavy chain variable region shown in SEQ ID NO: 6 and a light chain variable region shown in SEQ ID NO: 10. The antibody can specifically bind to Aβ(1-42) and is suitable for various immunoassays and research purposes.

[0011] In certain embodiments, the present invention further provides a monoclonal antibody against the Alzheimer's disease protein marker Aβ(1-42), wherein the monoclonal antibody is 3A8. The heavy chain variable region of monoclonal antibody 3A8 comprises H-CDR1 shown in SEQ ID NO: 15, H-CDR2 shown in SEQ ID NO: 16, and H-CDR3 shown in SEQ ID NO: 17, and the light chain variable region comprises L-CDR1 shown in SEQ ID NO: 19, L-CDR2 shown in SEQ ID NO: 20, and L-CDR3 shown in SEQ ID NO: 21.

[0012] In certain embodiments, the monoclonal antibody 3A8 consists of a heavy chain variable region shown in SEQ ID NO: 14 and a light chain variable region shown in SEQ ID NO: 18. The 3A8 antibody also has high specificity and can be used in a variety of Aβ(1-42)-related analysis and detection methods.

[0013] Finally, the present invention provides a sandwich ELISA detection kit for detecting human amyloid protein Aβ(1-42). The kit comprises a detection antibody and a capture antibody, wherein the detection antibody is selected from monoclonal antibody 3A8, and the capture antibody is selected from monoclonal antibody 2B6. Through this combination, the kit can provide sensitive and specific detection performance, and is suitable for quantitative detection of Aβ(1-42) in clinical and laboratory environments.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects:

[0015] The present invention discovers the Alzheimer's disease protein marker Aβ(1-42), and designs and reconstructs the immune antigen-enhanced Alzheimer's disease combined antigen such as SEQ ID NO: 1 by predicting the B cell epitope of the template Aβ(1-42) protein through ABCpred, which is referred to as AD-Aβ2.

[0016] Based on the antigen, monoclonal antibodies 2B6 and 3A8 with strong affinity and high titer were prepared. At the same time, a sandwich ELISA kit for detecting human amyloid protein Aβ based on 2B6 and 3A8 antibodies was provided, achieving an Aβ concentration detection line effect of at least 0.5 ng / mL, with strong specificity and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 qPCR identification of the Alzheimer's disease protein marker Aβ(1-42).

[0018] Figure 2 Screening of multi-epitope antigen sequences of Aβ(1-42).

[0019] Figure 3 Analysis of the tertiary structure of AD-Aβ2 monomer and the secondary structure of AD-Aβ2.

[0020] Figure 4 AKTA purification and SDS-PAGE electrophoresis of AD-Aβ2 recombinant protein.

[0021] Figure 52. Subtype detection of B6 and 3A8 monoclonal antibodies.

[0022] Figure 6 Determination of the titer of anti-AD-Aβ2 monoclonal antibodies.

[0023] Figure 7Sandwich ELISA assay based on 2B6 and 3A8 monoclonal antibodies. DETAILED DESCRIPTION

[0024] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0025] Example 1: Identification of Alzheimer's disease protein marker Aβ(1-42)

[0026] According to the records of the prior art, the protein marker Aβ(1-42) of Alzheimer's disease was screened, and peripheral blood samples (PB-AD Samples) of 40 patients with Alzheimer's disease and 40 healthy peripheral blood control samples (PB-HCSamples) were collected; 3 mL of whole blood was taken from all samples, added into EDTA anticoagulant tubes, plasma was separated and peripheral blood mononuclear cells (PBMCs) were extracted; according to the information of Chain A, Alzheimer's d isace amyloid, PDB:1IYT_A / Aβ(1-42):DAEFRHDSGYEVHHQKLVFFAED VGSNKGAIIGLMVGGVVIA (SEQ ID NO:1), specific primers for Aβ(1-42) were designed, and primer design and synthesis services were provided by Shanghai Biotech Co., Ltd., and then qPCR detection of Aβ(1-42) was performed on patients and healthy subjects using PCR Kit with Taq (PCR kit, containing Taq enzyme) provided by Biotech, see Figure 1 .

[0027] Figure 1 The results showed that the expression level of Aβ(1-42) in peripheral blood samples of Alzheimer's disease patients was significantly higher than that in healthy peripheral blood samples, that is, Aβ(1-42) can be used as a protein marker for Alzheimer's disease.

[0028] Example 2: Construction and analysis of the multi-epitope antigen of Aβ, a protein marker of Alzheimer's disease, based on bioinformatics

[0029] The sequence information of human Alzheimer's disease protein marker amyloid β (Aβ) was retrieved using the NCBI database, and the Aβ protein sequence was selected as follows: Chain A, Alzheimer's disease amyloid, PDB: 1IYT_A / Aβ(1-42): DAEFRHDSGYEVHHQKLVFFAEDVGSNK GAIIGLMVGGVVIA (SEQ ID NO: 1).

[0030] ABCpred was used to predict the B cell epitopes of the template Aβ(1-42) protein, with a threshold of 0.51. The differences between the Aβ protein sequence and the predicted B cell epitopes were compared, and the highly conserved epitope sequences located on the surface of the protein structure were predicted. Figure 2 .

[0031] Figure 2 The results showed the multi-epitope antigen sequence of Aβ(1-42), wherein Aβ(1-42)-1: FRHDSGYEVHHQKLVF (SEQ ID NO: 2); Aβ(1-42)-2: EVHHQKLVFFAEDVGS (SEQ ID NO: 3); Aβ(1-42)-3: SNKGAIIGLMVGGVVI (SEQ ID NO: 4).

[0032] The above sequences were connected in sequence using the flexible linker "SGGGGS" as a linker to construct a recombinant Aβ2 multi-epitope antigen sequence, namely [Aβ(1-42)-1-linker-Aβ(1-42)-2-linker-Aβ(1-42)-3-linker]6, named "Alzheimer's disease combined antigen", abbreviated as AD-Aβ2, and its amino acid sequence is as follows:

[0033] FRHDSGYEVHHQKLVFSGGGGSEVHHQKLVFFAEDVGSSGGGGSNKGAIIGLMVGGVVISGGGGSFRHDSGYEVHHQKLVFSGGGGSEVHHQKLVFFAEDVGSSGGGGSSNKGAIIGLMVGGVVISGGGGSFRHDSGYEVHHQKLVFSGGGGSEVHHQKLVFFAEDVGSSGGGGSSNKGAIIGLMVGGVVISGGGGSFR HDSGYEVHHQKLVFSGGGGSEVHHQKLVFFAEDVGSSGGGGSSNKGAIIGLMVGGVVISGGGGSFRHDSGYEVHHQKLVFSGGGGSEVHHQKLVFFAEDVGSSGGGGSSNKGAIIGLMVGGVVISGGGGSFRHDSGYEVHHQKLVFSGGGGSEVHHQKLVFFAEDVGSSGGGGSSNKGAIIGLMVGGVVISGGGGS(SEQ ID NO:5);

[0034] The tertiary structure of Aβ(1-42)-1-linker-Aβ(1-42)-2-linker-Aβ(1-42)-3-linker monomer was constructed using SWISS-MODEL. The secondary structure of AD-Aβ2 was analyzed using the novopro online tool. Figure 3 .

[0035] Figure 3 The results showed that the tertiary structure constructed by SWISS-MODEL can intuitively demonstrate that the antigenic epitopes of AD-Aβ2 are evenly distributed, fully exposed, structurally rigid, and reasonably arranged in space. The protein secondary structure prediction by novopro showed the presence of a large number of α-helices, β-folds, or irregular curls, that is, the AD-Aβ2 antigen peptide has richer antigenic epitopes than Aβ(1-42).

[0036] Example 3. Preparation and identification of recombinant protein of AD-Aβ2

[0037] The recombinant plasmid pET-28a-AD-Aβ2 with a His tag was constructed by BGI, and the recombinant plasmid pET-28a-AD-Aβ2 was transformed into Escherichia coli DH5α competent cells and plated for overnight culture. The positive clones were picked for sequencing the next day, and the plasmids extracted from the positive bacterial solution with correct sequencing were transformed into BL21 competent cells. The positive cells were picked and inoculated into LB medium and shaken at 37°C overnight. 1% fresh LB liquid medium was inoculated with bacteria, shaken at 37°C, and when the OD600 of the bacterial solution was about 0.5, IPTG was added to induce expression, and the bacteria were harvested after 4 hours. After ultrasonic disruption, centrifugation was performed to obtain inclusion body protein bacteria, which were then resuspended with 20mmol / LTris-HCl (pH=8.0), ultrasonically disrupted, dissolved with 8mol / L urea buffer, and centrifuged to collect the supernatant. After filtration, it was purified with a Ni-NTA affinity chromatography column, washed with 100mmol / L imidazole and 300mmol / L imidazole, and the eluate was collected and placed in a dialysis bag. It was stirred and renatured with urea buffer of different concentration gradients at 4°C, and the dialysate was replaced once every 6 hours for a total of 3 times to obtain the renatured AD-Aβ2 recombinant protein, which was then further purified with an AKTA protein purifier, and the molecular weight was detected by SDS-PAGE electrophoresis. Figure 4 .

[0038] Figure 4 The results showed that the specific target band of AD-Aβ2 recombinant protein had a high separation purity and the molecular weight was consistent with the expected 39.16kDa.

[0039] Example 4. Preparation and identification of anti-AD-Aβ2 monoclonal antibodies

[0040] BALB / c mice were immunized with equal volumes of AD-Aβ2 recombinant protein and Freund's adjuvant, with 50 μg of antigen per mouse, and immunized once every 14 days. After immunization 3 times, AD-Aβ2 protein was used as the detection antigen to detect the titer of mouse serum. The collected immune mouse serum was diluted in multiples, and 100 μl was added to each well of the AD-Aβ2 antigen-coated plate, incubated at 37°C for 30 minutes, washed 5 times with PBST, and patted dry. 100 μl of rabbit anti-mouse IgG-HRP (1:1500) was added to each well, incubated at 37°C for 30 minutes, washed 5 times with PBST, and patted dry. 50 μl of colorimetric solution A and solution B were added, and incubated at 37°C for 20 minutes. 50 μl of stop solution was added to each well. Reading was performed using a full-wavelength microplate reader (450nm, 630nm). Cell fusion was performed when the titer reached 1:8000, and cultured with HAT selection medium, and replaced with HT medium after 7 days. After 3 days, the hybridoma cell supernatant was coated with AD-Aβ2 recombinant protein and tested. Subcloning was performed twice by limiting dilution using HT medium, and finally two cell lines 2B6 and 3A8 that could stably secrete antibodies were screened. The hybridoma ascites was further prepared by expanding the culture. 10 BALB / c were taken, and 500 μL of Freund's incomplete adjuvant was injected into the peritoneum of each in advance. After 7 days, the hybridoma cell suspension was injected into the peritoneum of each mouse. 1×10 6 After the mouse abdomen was bulged, the ascites was collected and the antibodies were purified by saturated ammonium sulfate precipitation. The cell supernatants of 2B6 and 3A8 were collected and the subtypes of the monoclonal antibodies were tested using an antibody subtype detection kit. Figure 5 .

[0041] Figure 5 The results showed that subtype determination showed that 2B6 was IgG2a and 3A8 was IgG2b.

[0042] Total RNA was extracted from 2B6 and 3A8 monoclonal antibody hybridoma cells, respectively. RNA was reverse transcribed into cDNA using a reverse transcription kit. This step ensures that the VH and VL gene regions can be effectively transcribed into templates. Primers were designed based on the framework sequences of the conserved regions of the immunoglobulin heavy chain (VH) and light chain (VL) of mouse IgG2. The VH and VL gene regions were PCR amplified. The PCR products were then detected using 1.5%-2% agarose gel electrophoresis to observe whether the VH and VL bands were as expected (about 350–400 bp). The target bands were recovered and purified using a PCR product purification kit. The PCR products were cloned into a plasmid vector and subsequently sequenced. The purified PCR products were ligated to pGEM-T, transformed into competent E. coli cells, screened for positive clones, and plasmid DNA was extracted. The plasmids were sent to Novogene Sequencing for bidirectional sequencing. The measured VH and VL sequences were submitted to the immunoglobulin gene database (IgBLAST or IMGT) for sequence comparison, the sequences of their variable regions (FR and CDR regions) were identified, and the CDR1, CDR2 and CDR3 sequences were determined (see Table 1).

[0043] Table 1 Identification of variable region sequences of 2B6 and 3A8 monoclonal antibodies

[0044]

[0045]

[0046] Example 5. Determination of the titer of anti-AD-Aβ2 monoclonal antibodies

[0047] AmyloidβPeptide(1-42)human commercial Aβ standard was provided by Biotech. The Aβ standard was diluted and added to a 96-well ELISA plate and incubated overnight at 4°C; then 5% skim milk was added for blocking and incubated at 37°C for 1h; the purified ascites was quantified and diluted to 1mg / mL, and added to the ELISA plate in multiple dilutions starting from 1:2000 as the primary antibody, and mouse positive and negative serum was used as the positive and negative control, incubated at 37°C for 1h; HRP-sheep anti-mouse secondary antibody was diluted 1:8000 with 1×PBS and added to the ELISA plate, incubated at 37°C for 30min; finally, TMB was added for color development for 8min, and then the stop solution was added to terminate the reaction, and the OD450 was measured with an ELISA instrument. The dilution corresponding to the well with the OD450 of the sample to be tested / OD450 of the negative sample (S / N)>2.1 was the ascites titer, see Figure 6 .

[0048] Figure 6The results showed that when the purified 2B6 monoclonal antibody was diluted to a concentration of 1 mg / mL and then further diluted in multiples, the antibody titer was at least 1:32000; the titer of 3A8 was determined by the same method, and the results showed that when the monoclonal antibody concentration was 1 mg / mL, the antibody titer was also at least 1:32000, indicating that 2B6 and 3A8 can well recognize human amyloid protein Aβ and have very high titers.

[0049] Example 6: Sandwich ELISA to detect human amyloid protein Aβ

[0050] Add 2B6 monoclonal antibody (diluted to 2μg / mL, PBS solution) to the 96-well ELISA plate, add 100μL to each well, incubate at 4℃ overnight, pour out the unbound capture antibody 2B6 solution, wash 3 times (PBS+0.05% Tween-20), add 200μL 1% BSA blocking solution to each well, incubate at 37℃ for 1 hour to reduce nonspecific adsorption, remove the blocking solution, add different concentrations of commercial Aβ standard antigen (0.5, 1, 2, 5, 10ng / mL) provided by Beyotime Biotechnology to each well, 100μL per well. Incubate at 37℃ for 1 hour to promote Aβ antigen binding to capture antibody 2B6. Wash 3 times with washing solution after incubation. HRP-labeled 3A8 detection monoclonal antibody (diluted to 1 μg / mL, PBS solution) was added to each well, 100 μL per well; incubated at 37°C for 1 hour to promote the formation of antigen-antibody complex, and washed 5 times with washing solution after incubation; 100 μL TMB colorimetric solution was added to each well, and reacted at room temperature in the dark for 10-15 minutes. The reaction time was controlled at the stage where the color development was significant but not excessive, and 50 μL stop solution (1MH2SO4) was added to each well to terminate the color development reaction, and the absorbance (OD value) was read at a wavelength of 450 nm. The standard curve was drawn with the Aβ antigen concentration (ng / mL) as the horizontal axis and the absorbance (OD450 value) as the vertical axis, and the curve correlation analysis between absorbance and Aβ protein concentration was established, see Figure 7 .

[0051] The fitting formula is as follows: y = 0.31 × ln (x) + 0.29

[0052] y: absorbance (OD450)

[0053] x: Aβ concentration (ng / mL)

[0054] Figure 7The results showed that the absorbance (OD450) was logarithmically related to the Aβ concentration, which was consistent with the absorbance characteristics of the enzyme reaction and the antibody binding kinetics in the sandwich ELISA. At low concentrations (0.5-2ng / mL), the absorbance increased rapidly with increasing concentrations. In the higher concentration range (5-10ng / mL), the increase in absorbance tended to slow down, which was consistent with the saturation effect of the enzyme reaction. The experimental data points (blue dots) were very close to the fitting curve (red line), and the fitting degree was high, indicating that the experimental data were credible and the determination method was stable. The fitting consistency was good in the low concentration range (0.5-2ng / mL) and the high concentration range (5-10ng / mL). The sandwich ELISA method based on monoclonal antibodies 2B6 and 3A8 had a good linear correlation in the Aβ concentration range of 0.5-10ng / mL, and the absorbance and concentration conformed to the logarithmic model, which could reliably detect Aβ antigen quantitatively.

[0055] 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. An Alzheimer's disease protein marker, characterized in that: The marker is the Aβ(1-42) protein as shown in SEQ ID NO:

1.

2. An antigen peptide, characterized in that The antigenic peptide consists of the antigenic epitope peptide of the Alzheimer's disease protein marker Aβ(1-42) shown in SEQ ID NO:2-4.

3. The antigen peptide according to claim 2, characterized in that: The amino acid sequence of the antigen peptide is shown in SEQ ID NO:

5.

4. A monoclonal antibody against Aβ(1-42), a protein marker for Alzheimer's disease, characterized in that: The monoclonal antibody is 2B6, wherein the heavy chain variable region of monoclonal antibody 2B6 consists of H-CDR1 shown in SEQ ID NO:7, H-CDR2 shown in SEQ ID NO:8 and H-CDR3 shown in SEQ ID NO:9, and its light chain variable region consists of L-CDR1 shown in SEQ ID NO:11, L-CDR2 shown in SEQ ID NO:12 and L-CDR3 shown in SEQ ID NO:

13.

5. The monoclonal antibody according to claim 4, characterized in that: The monoclonal antibody 2B6 consists of a heavy chain variable region represented by SEQ ID NO:6 and a light chain variable region represented by SEQ ID NO:

10.

6. A monoclonal antibody against Aβ(1-42), a protein marker for Alzheimer's disease, characterized in that: The monoclonal antibody is 3A8, wherein the heavy chain variable region of monoclonal antibody 3A8 consists of H-CDR1 shown in SEQ ID NO:15, H-CDR2 shown in SEQ ID NO:16 and H-CDR3 shown in SEQ ID NO:17, and its light chain variable region consists of L-CDR1 shown in SEQ ID NO:19, L-CDR2 shown in SEQ ID NO:20 and L-CDR3 shown in SEQ ID NO:

21.

7. The monoclonal antibody according to claim 6, characterized in that The monoclonal antibody 3A8 consists of a heavy chain variable region represented by SEQ ID NO:14 and a light chain variable region represented by SEQ ID NO:

18.

8. A sandwich ELISA detection kit for detecting human amyloid protein Aβ(1-42), the kit comprising a detection antibody and a capture antibody, wherein the detection antibody is selected from the monoclonal antibody 3A8 described in claim 6 or 7, and the capture antibody is selected from the monoclonal antibody 2B6 described in claim 4 or 5.

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