Antibody kit for detecting Alzheimer disease and application thereof

Abstract

The invention relates to the technical field of biomedical detection, and particularly discloses an antibody kit for detecting Alzheimer's disease and application of the antibody kit. The kit comprises a coated antibody 4H2, an enzyme-labeled antibody 5H4 and an A beta (1-42) antigen standard substance, wherein the 4H2 and the 5H4 are respectively anti-A beta (1-42) monoclonal antibodies for identifying different sites. Specific detection of A beta (1-42) is achieved through a double-antibody sandwich method, good sensitivity and specificity are achieved, the lowest limit of detection (LOD) is 1.34 ng / mL, and the kit is suitable for auxiliary diagnosis of the early Alzheimer's disease. The antibody can stably recognize A beta (1-42), and is suitable for development of kits, automatic detection platforms and related clinical detection reagents.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to an antibody kit for detecting Alzheimer's disease and its application. Background Technology

[0002] Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by cognitive impairment. Its pathogenesis is complex, and there is currently no cure. Research indicates that amyloid-β (Aβ), especially the Aβ(1-42) peptide, is a core factor in the early pathological changes of AD. Its abnormal accumulation in the brain can form plaques and exert toxicity on neurons. Therefore, Aβ(1-42) concentration has become an important biomarker for the early diagnosis and assessment of AD disease progression.

[0003] Existing methods for detecting Aβ(1-42) mainly include Western blot, immunoblotting, electrochemical methods, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Among these, ELISA is widely used in preclinical research and in vitro diagnostics due to its high throughput, high sensitivity, and moderate cost. However, the antibodies used in traditional ELISA are mostly derived from polyclonal antibodies or low-affinity monoclonal antibodies, which have problems such as poor specificity, insufficient stability, and high background. In particular, it is difficult to distinguish Aβ(1-42) from homologous peptides with similar structures, such as Aβ40, which affects the accuracy of detection.

[0004] To address the aforementioned issues, there is an urgent need to develop a dual-antibody combination with good specificity and high affinity to construct a stable, efficient, and reproducible sandwich ELISA kit. This invention designs and expresses the Aβ(1-42) trimer antigen fragment, immunizes mice to obtain high-affinity monoclonal antibodies 4H2 and 5H4 that recognize different sites, and establishes a dual-antibody sandwich method with a sensitivity of 1.34 ng / mL, enabling highly specific detection of Aβ(1-42), suitable for early AD auxiliary diagnosis and related research scenarios. Summary of the Invention

[0005] The purpose of this invention is to provide an antibody kit and detection method for detecting the Alzheimer's disease biomarker Aβ(1-42), which can achieve high specificity and high sensitivity detection of Aβ(1-42), thereby providing strong technical support for the early screening and auxiliary diagnosis of Alzheimer's disease.

[0006] To achieve the above objectives, the present invention provides an antibody kit comprising: a first monoclonal antibody 4H2 coated on a solid-phase support, wherein the amino acid sequences of the complementarity-determining regions (CDRs) of its heavy and light chain variable regions are as follows: heavy chain CDR1: SEQ ID NO:5, heavy chain CDR2: SEQ ID NO:6, heavy chain CDR3: SEQ ID NO:7; light chain CDR1: SEQ ID NO:9, light chain CDR2: SEQ ID NO:10, light chain CDR3: SEQ ID NO:11; and a second monoclonal antibody 5H4 labeled with horseradish peroxidase (HRP) that binds to Aβ(1-42), wherein the amino acid sequences of the CDRs of its heavy and light chain variable regions are as follows: heavy chain CDR1: SEQ ID NO:13, heavy chain CDR2: SEQ ID NO:14, heavy chain CDR3: SEQ ID NO:15;

[0007] Light chain CDR1: SEQ ID NO:17, light chain CDR2: SEQ ID NO:18, light chain CDR3: SEQ ID NO:19; Aβ(1-42) antigen standard, preferably a recombinant antigen expressed in the form of trimer (Aβ42)3; auxiliary reagents such as enzyme substrate solution (e.g., TMB), stop solution (e.g., 2M sulfuric acid), wash buffer (e.g., PBST) and blocking solution (e.g., BSA solution).

[0008] Preferably, the 4H2 and 5H4 antibodies recognize different non-overlapping epitopes in the Aβ(1-42) molecule, respectively, without cross-interference, making them suitable for sandwich assay applications. According to SPR detection, the affinity constants (KD) of both antibodies with Aβ(1-42) are 4.0 × 10⁻⁶. -9 M and 1.4×10 -8 M has high affinity.

[0009] The present invention also provides a method for detecting Aβ(1-42) based on the above-mentioned kit, comprising the following steps:

[0010] Coat the first antibody 4H2 onto the ELISA plate; add the sample to be tested or the standard, incubate and wash; add the HRP-labeled second antibody 5H4; after the colorimetric reaction, add the stop solution; read the absorbance at 450 nm wavelength, and calculate the Aβ(1-42) concentration using the standard curve.

[0011] Compared with the prior art, the beneficial effects of the present invention include: the dual antibody combination provided by the present invention has a clear epitope recognition, high affinity and strong stability, and is suitable for standardized industrial production; the reagent kit is easy to operate and can be used for quantitative detection of Aβ(1-42) in various samples such as serum and cerebrospinal fluid; the detection method has high sensitivity and is suitable for early screening and clinical auxiliary diagnosis of Alzheimer's disease. Attached Figure Description

[0012] Figure 1 SDS-PAGE detection of the (Aβ42)3 recombinant antigen fragment.

[0013] Figure 2 Positive clone screening results for monoclonal antibodies against the (Aβ42)3 recombinant antigen fragment.

[0014] Figure 3 Detection of monoclonal antibody affinity kinetic constants based on surface plasmon resonance (SPR) technology.

[0015] Figure 4 Linear regression equation and correlation coefficient for Aβ(1-42) detection using a double-antibody sandwich method. Detailed Implementation

[0016] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0017] Example 1: Preparation of the (Aβ42)3 recombinant antigen fragment

[0018] According to NCBI records, ChainA, Alzheimer's disease amyloid, PDB: 1IYT_A / Aβ(1-42): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 1), a redesigned (Aβ42)3 recombinant antigen fragment (Aβ42-linker-Aβ42-linker-Aβ42), abbreviated as (Aβ42)3, has the following amino acid and nucleotide sequences:

[0019] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAGGGGS

[0020] GGGGSGGGGSDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV

[0021] IAGGGGSGGGGSGGGGSDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL

[0022] MVGGVVIA (SEQ ID NO:2);

[0023] gatgcggaatttcgccatgatagcggctatgaagtgcatcatcagaaactggtgttttttgcggaagatgtgggcagcaacaaaggcgcgattattggcctgatggtgggcggcgtgg tgattgcgggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcgatgcggaatttcgccatgatagcggctatgaagtgcatcatcagaaactggtgttttttgcgga agatgtgggcagcaacaaaggcgcgattattggcctgatggtgggcggcgtggtgattgcgggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcgatgcggaattt cgccatgatagcggctatgaagtgcatcatcagaaactggtgttttttgcggaagatgtgggcagcaacaaaggcgcgattattggcctgatggtgggcggcgtggtgattgcg(SEQ ID NO:3);

[0024] The (Aβ42)3 gene fragment was synthesized by Suzhou Genewise Biotech Co., Ltd., and ligated into the pET-28a vector containing 6 histidine residues to construct the pET-28a-(Aβ42)3 recombinant expression plasmid. The expression plasmid pET-28a-(Aβ42)3 was transformed into E. coli BL21(DE3) competent cells. After PCR detection, positive colonies were picked and inoculated into LB liquid medium (containing 50 μg·μL). -1 In kanamycin, the culture was incubated at 37°C with shaking for 12 hours; an appropriate amount of the bacterial culture was then transferred to a new culture medium, and the OD600 was adjusted to 0.2. The culture was then incubated at 37°C and 220 rpm until the OD600 reached 0.4-0.6; IPTG was then added to a final concentration of 1 mmol / L. -1The cells were induced and cultured at 37℃ for 16-20 h. After induction, the bacterial culture was centrifuged at 4℃ and 4000×g, and the precipitate was collected. The cells were resuspended in phosphate buffer, sonicated on ice for 30 min, and then centrifuged at 4℃ and 8500×g for 15 min. The supernatant and precipitate were collected, and protein expression was detected by SDS-PAGE. SDS-PAGE analysis showed that the (Aβ42)3 recombinant protein was mainly present in the supernatant. The supernatant was collected, filtered through a 0.45 μm filter membrane, and added to a nickel column. The target protein was eluted with equilibration buffer containing different concentrations of imidazole (20, 100, 200, 300 mmol / L). Each eluted fraction was collected for SDS-PAGE analysis. Figure 1 .

[0025] Figure 1 The results showed that the molecular weight of the (Aβ42)3 recombinant protein was 15.40 kDa, and its purity met expectations, making it suitable for subsequent immunization of mice.

[0026] Example 2: Preparation and Identification of Monoclonal Antibodies

[0027] Five BALB / c mice were immunized with the (Aβ42)3 recombinant protein at a concentration of 1 mg / mL, divided into four doses. For the first three immunizations, 50 μL of protein solution was mixed with an equal volume of Freund's adjuvant and injected subcutaneously at multiple sites. Seven days after the third immunization, serum was collected from the tail vein. Antibody titers in the serum were determined by indirect ELISA. Serum from unimmunized mice served as a negative control. Mice with the highest antibody response were given a pulse immunization with 100 μL of (Aβ42)3 antigen protein injected intraperitoneally. Three days after the pulse immunization, the mice were dissected, and spleen cells were fused with SP2 / 0 myeloma cells. Positive hybridoma cells were screened using indirect ELISA. SP2 / 0 cell culture supernatant was used as a negative control. Positive wells were enlarged for culture, and strongly positive wells were subcloned using limiting dilution, with cells cryopreserved during the process. After three subclonings, hybridoma cell lines with high and stable positive values ​​were selected as the final positive hybridoma cell lines. Figure 2 .

[0028] Figure 2 The results showed that the hybridoma cell lines 4H2 and 5H4 had higher positive values, and further identification was carried out.

[0029] Twenty 6-8 week old BALB / c mice were prepared and injected intraperitoneally with sterile liquid paraffin (500 μL / mouse) one week prior to injection. The prepared positive hybridoma cell lines 4H2 and 5H4 were then injected at 1 × 10⁻⁶ mg / mL. 6Inject only into the peritoneal cavity of mice; 14 days later, when the mice's abdomens were significantly distended and their mental state was poor, begin to gradually extract ascites; use a Protein L antibody purification kit to purify the monoclonal antibodies 4H2 and 5H4 against the (Aβ42)3 antigen fragment in the mouse ascites, and then extract the supernatant of the hybridoma cell line (at a ratio of 1:2, 1:2...). 2 1:2 3 1:2 4 To 1:2 12 (diluted) and mouse ascites (at 1:10, 1:10) 2 1:10 3 1:10 4 1:10 5 To 1:10 8 Antibody titers (diluted) were detected by indirect ELISA, as shown in Table 1.

[0030] Table 1 Results of antibody sensitivity assay by indirect ELISA

[0031]

[0032] Table 1 shows that the antibody titers in the supernatant of cells from strains 4H2 and 5H4 reached 1:2. 10 1:2 9 The antibody titer in the ascites fluid of 4H2 mice reached 1:10. 8 The antibody titer in the ascites fluid of 5H4 mice can reach 1:10. 7 .

[0033] The affinity kinetics of monoclonal antibodies were determined using surface plasmon resonance (SPR) technology: EDC / NHS mixture (1:1 volume ratio) was injected at a flow rate of 10 μL / min for 7 min to activate the carboxyl groups on the chip surface. Aβ(1-42) (purchased from Jier Biochemical (Shanghai) Co., Ltd.) was dissolved in 10 mM sodium acetate buffer (pH 4.5) at a concentration of 20 μg / mL and injected into the chip to optimize the coupling density. 1M ethanolamine (pH 8.5) was injected for 7 min to block residual active groups. 4H2 and 5H4 were diluted to five concentration gradients (0.78 nM to 100 nM), with each concentration repeated three times. The antibody solution was injected at a flow rate of 30 μL / min for 120 s, and the binding signal (RU value) was monitored in real time. The solution was then switched to the run buffer, and dissociation was monitored for 240 s. Inject 10 mM glycine-HCl (pH 1.5) for 30 s to restore baseline and ensure signal stability. Affinity constant (KD): Calculate KD = koff / kon, see [link to relevant documentation]. Figure 3 .

[0034] Figure 3The results showed that the dissociation constants (Kd) of 4H2 and 5H4 were 4.0 × 10⁻⁶ and 4.0 × 10⁻⁶, respectively. -9 and 1.4×10 -8 M, the results showed that 4H2 and 5H4 both had good affinity for Aβ(1-42).

[0035] Identification of the heavy and light chain variable region sequences of monoclonal antibodies 4H2 and 5H4:

[0036] Take 2 × 10⁻⁶ hybridoma cells of 4H₂ and 5H₄ in the logarithmic growth phase. 6 Total RNA was extracted using TRIzol and other methods; concentration and purity were determined using Nanodrop (A260 / A280 was 1.8-2.0); 1 μg of RNA was used for reverse transcription. First-strand cDNA reverse transcription was performed using Oligo(dT) primers or Random Hexamer; a high-efficiency reverse transcriptase (SuperScript III) was used to ensure complete reverse transcription of the V region. PCR amplification was performed using Mouse IgG-specific universal primers (or specific primers for IgG1, Igκ / λ); PCR conditions: 98℃ pre-denaturation for 30 s, 98℃ for 10 s → 60℃ for 20 s → 72℃ for 30 s, 30 cycles, and a final extension at 72℃ for 5 minutes; the PCR product was purified by gel extraction and ligated into a T / A cloning vector; DH5α competent cells were transformed, and single clones were selected; samples were sent for sequencing or bidirectional sequencing was performed using a Sanger sequencer; at least 3 positive clone sequences were selected to confirm identity; the CDR1 / 2 / 3 structure was analyzed; the frame regions (FRs) were labeled and encoded according to kabat.

[0037] Table 2. Sequence analysis of the heavy and light chain variable regions of monoclonal antibodies 4H2 and 5H4.

[0038]

[0039]

[0040]

[0041] Example 3: Double Antibody Sandwich Method for Detecting Aβ (1-42)

[0042] Dilute 4H2 antibody to 2 μg / mL with coating buffer, add 100 μL to each well, and incubate overnight (16 hours) at 4°C. Discard the liquid and wash three times with PBST (200 μL each time, incubate for 1 minute). Add 200 μL of blocking buffer to each well and block at 37°C for 2 hours. Discard the blocking buffer and wash three times with PBST. Dilute Aβ(1-42) antigen standard (purchased from Jier Biochemical (Shanghai) Co., Ltd.) with PBS containing 0.1% BSA (range 1.34-100 ng / mL). Add 100 μL of antigen solution to each well and incubate at 37°C for 1 hour. Wash five times with PBST (to reduce non-specific binding). Dilute 5H4-HRP antibody to 0.5 μg / mL with blocking buffer, add 100 μL to each well, and incubate at 37°C for 45 minutes. Wash five times with PBST. Add 100 μL of TMB substrate solution to each well and react at room temperature in the dark for 15 minutes. Add 50 μL of stop solution (2M sulfuric acid) to terminate the reaction. Immediately measure the absorbance (OD value) at 450 nm using a microplate reader. Plot a curve with antigen concentration on the x-axis and OD value on the y-axis, see [reference needed]. Figure 4 .

[0043] Figure 4 The results showed that the sensitivity of the double-antibody sandwich method for detecting Aβ(1-42) was at least 1.34 ng / mL.

[0044] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antibody kit for detecting Alzheimer's disease markers, characterized in that: The kit comprises: a first monoclonal antibody 4H2 coated on a solid phase carrier; the amino acid sequences of the complementary determining regions (CDRs) of its heavy and light chain variable regions are as follows: heavy chain CDR1: SEQ ID NO: 5, heavy chain CDR2: SEQ ID NO: 6, heavy chain CDR3: SEQ ID NO: 7; light chain CDR1: SEQ ID NO: 9, light chain CDR2: SEQ ID NO: 10, light chain CDR3: SEQ ID NO: 11; a second monoclonal antibody 5H4 labeled with horseradish peroxidase (HRP); the amino acid sequences of the CDRs of its heavy and light chain variable regions are as follows: heavy chain CDR1: SEQ ID NO: 13, heavy chain CDR2: SEQ ID NO: 14, heavy chain CDR3: SEQ ID NO: 15; light chain CDR1: SEQ ID NO: 17, light chain CDR2: SEQ ID NO: 18, light chain CDR3: SEQ ID NO:

19. NO: 19; Aβ(1-42) antigen standard; enzyme substrate solution, stop solution, washing buffer and blocking solution.

2. The antibody kit according to claim 1, characterized in that The first antibody 4H2 and the second antibody 5H4 respectively have the following variable region sequences: the 4H2 heavy chain variable region sequence is such as SEQ ID NO: 4, and the light chain variable region sequence is such as SEQ ID NO: 8; the 5H4 heavy chain variable region sequence is such as SEQ ID NO: 12, and the light chain variable region sequence is such as SEQ ID NO:

16.

3. A method for detecting Aβ(1-42) based on the kit according to any one of claims 1 to 2, comprising the following steps: Coat 4H2 on an ELISA plate; add the sample to be tested, incubate, and then wash; add HRP-labeled 5H4 antibody; add stop solution after color development; read the absorbance at a wavelength of 450nm and analyze the results.

4. The method according to claim 3, characterized in that The limit of detection (LOD) of the method was 1.34 ng / mL.

5. Use of the kit according to any one of claims 1 to 2 in the preparation of an in vitro detection reagent for detecting Aβ(1-42) in a biological sample to assist in the early diagnosis of Alzheimer's disease.

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