Monoclonal antibody combination for simultaneously detecting apo e2 / 3 / 4 proteins and application thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody combination for simultaneously detecting APOE2 / 3 / 4 proteins and application thereof. The combination comprises monoclonal antibody 3H7 and monoclonal antibody 2C8, can simultaneously recognize three protein isomers of APOE2 / 3 / 4 with high specificity and high sensitivity, and is suitable for rapid detection reagent development. The monoclonal antibody 3H7 and the monoclonal antibody 2C8 respectively have a clearly defined complementarity determining region sequence, and the CDR amino acid sequences of the heavy chain and light chain variable regions are respectively SEQ ID NO. 1-6 and SEQ ID NO. 7-12. A colloidal gold immunochromatography test strip rapid detection tool is constructed in the application, the detection sensitivity can reach 0.5 ng / mL, and the application has extremely high specificity, thereby providing an efficient and reliable solution for clinical screening and disease risk assessment of APOE proteins.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a combination of monoclonal antibodies that can simultaneously detect APOE2 / 3 / 4 proteins and their applications. Background Technology

[0002] Apolipoprotein E (APOE) is a polymorphic protein with important biological significance and clinical diagnostic value. It can regulate lipoprotein metabolism and cholesterol transport in the cardiovascular and central nervous systems, and also plays an important role in the pathogenesis of Alzheimer's disease (AD), such as β-amyloid (Aβ) deposition, tau protein hyperphosphorylation, and neuroinflammatory responses.

[0003] In humans, APOE consists of 299 amino acids with a molecular weight of 34 kD and has three main isoforms: APOE2, APOE3, and APOE4, encoded by the APOE α2, α3, and α4 alleles, respectively. The APOE gene exhibits polymorphism in the population, with significant differences in genotype among different populations. Variations in APOE alleles are considered to be associated with various diseases, including atherosclerosis, nephrotic syndrome, diabetes, and Alzheimer's disease. Besides gene polymorphism, plasma APOE protein levels also reflect disease status. APOE2 has a weak binding affinity to the low-density lipoprotein receptor (LDLR), leading to its association with high plasma cholesterol levels and becoming a risk factor for atherosclerosis. APOE4 can significantly increase total cholesterol concentration and plasma LDL levels in healthy individuals, accelerating subjective cognitive decline, memory loss, and the progression to mild cognitive impairment; it is considered a risk factor for cardiovascular disease and Alzheimer's disease. Studies have shown that peripheral blood APOE protein levels in Alzheimer's disease patients are lower than in healthy controls, and decreased plasma APOE protein levels are positively correlated with an increased risk of Alzheimer's disease. Even those with the lowest APOE protein levels still have a three-fold increased risk of Alzheimer's-related dementia compared to those with the highest levels. Therefore, lower APOE protein levels are considered a potential risk factor independent of the APOEα4 gene. The "Expert Consensus on the Standardized Application of APOEα4 Testing in Clinical Practice of Alzheimer's Disease" states that low peripheral blood APOE protein levels are independently associated with the long-term risk of sporadic Alzheimer's disease and recommends it as a biomarker for early screening of high-risk groups (Class IIa recommendation, Level B evidence). Low peripheral blood APOE protein levels have potential value in predicting the progression of mild cognitive impairment (Class IIb recommendation, Level C evidence). Therefore, peripheral blood APOE protein testing is of great significance for risk assessment and prediction of various diseases.

[0004] Current methods for detecting APOE protein in plasma and serum include radioimmunoassay and immunoturbidimetry. These methods generally suffer from problems such as complex operation, long processing times, and strong equipment dependence, making them unsuitable for the needs of primary healthcare institutions or rapid on-site screening. Furthermore, existing commercially available antibodies generally suffer from insufficient specificity for recognizing different APOE isoforms, strong cross-reactivity, or insufficient sensitivity, making it difficult to achieve unified and accurate detection of APOE2, APOE3, and APOE4 proteins. Especially in the development of rapid detection tools based on the double-antibody sandwich principle (such as colloidal gold immunochromatographic test strips), the lack of a pair of high-affinity, high-specificity monoclonal antibodies capable of simultaneously recognizing all three isoforms severely restricts the standardization and widespread adoption of APOE protein detection technology. Summary of the Invention

[0005] In view of the above-mentioned shortcomings and deficiencies in the prior art, this application provides a monoclonal antibody combination for simultaneous detection of APOE2 / 3 / 4 proteins, which solves the technical problem of the lack of monoclonal antibody pairing that can simultaneously recognize three APOE isoforms with high specificity and high sensitivity and is suitable for the development of rapid detection reagents.

[0006] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0007] In a first aspect, this application provides a monoclonal antibody combination for simultaneous detection of APOE2 / 3 / 4 proteins, the monoclonal antibody combination comprising monoclonal antibody 3H7 and monoclonal antibody 2C8, wherein the heavy chain variable region of monoclonal antibody 3H7 includes three complementarity-determining regions, the amino acid sequences of which are shown as SEQ ID NO.1-SEQ ID NO.3 respectively;

[0008] The light chain variable region of the monoclonal antibody 3H7 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0009] The heavy chain variable region of the monoclonal antibody 2C8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively.

[0010] The light chain variable region of the monoclonal antibody 2C8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0011] In a further embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO. 14.

[0012] In a further embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2C8 is shown in SEQ ID NO. 15; the amino acid sequence of the light chain variable region of the monoclonal antibody 2C8 is shown in SEQ ID NO. 16.

[0013] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO. 18.

[0014] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2C8 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2C8 is shown in SEQ ID NO. 20.

[0015] Secondly, this application provides the application of the above-mentioned monoclonal antibody combination in the preparation of a monoclonal antibody tool for detecting APOE2 / 3 / 4 proteins.

[0016] In a further embodiment, the tool includes colloidal gold test strips, reagents, kits, and antibody chips.

[0017] In a further embodiment, the colloidal gold test strip uses monoclonal antibody 3H7 as the capture antibody and monoclonal antibody 2C8 as the labeling antibody.

[0018] In a further embodiment, the colloidal gold test strip includes a nitrocellulose membrane, a colloidal gold pad, a sample pad, and absorbent paper attached to a backing plate.

[0019] In a further embodiment, the nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 3H7, the control line includes goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 2C8.

[0020] Beneficial effects:

[0021] This invention provides a monoclonal antibody combination for the simultaneous detection of APOE2 / 3 / 4 proteins. This combination comprises monoclonal antibody 3H7 and monoclonal antibody 2C8, capable of simultaneously recognizing three protein isoforms—APOE2, APOE3, and APOE4—with high specificity and sensitivity, and is suitable for the development of rapid detection reagents. Monoclonal antibodies 3H7 and 2C8 each possess well-defined complementarity-determining regions (CDRs), with the CDR amino acid sequences of their heavy and light chain variable regions being SEQ ID NO. 1-6 (3H7) and SEQ ID NO. 7-12 (2C8), respectively. This invention further develops rapid detection tools such as colloidal gold immunochromatographic test strips. Results show that the detection sensitivity reaches 0.5 ng / mL, with no cross-reactivity with irrelevant proteins, exhibiting extremely high specificity. This provides a reliable tool for the efficient and accurate detection of APOE proteins, suitable for applications in related scientific research and bioanalysis fields, and also provides an efficient and reliable solution for clinical screening of APOE proteins and related disease risk assessment. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Image showing the results of SDS-PAGE protein identification;

[0024] Figure 2 The diagram shows the identification results of recombinant proteins APOE2 (A), APOE3 (B), and APOE4 (C).

[0025] Figure 3 This is a schematic diagram of colloidal gold assembly.

[0026] Figure 4 This is a graph showing the results of the specificity analysis of the paper strips;

[0027] Figure 5 This is a graph showing the sensitivity test results of the test strips;

[0028] Figure 6 The binding reaction of recombinant proteins APOE2 (left), APOE3 (middle), and APOE4 (right) with monoclonal antibodies. Detailed Implementation

[0029] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0030] These embodiments are provided to make the application thorough and complete, and to fully express the scope of the application to those skilled in the art. It should be noted that, unless otherwise specifically stated, the relative arrangement of components and steps, material composition, numerical expressions, and values ​​illustrated in these embodiments should be interpreted as merely exemplary and not as limiting.

[0031] APOE2 / 3 / 4 proteins include recombinant APOE2 / 3 / 4 proteins and native proteins.

[0032] Using the monoclonal antibody combination 3H7 and 2C8 protected in this application, APOE2, APOE3, and APOE4 can be detected individually. APOE2 / 3 or APOE2 / 4 and APOE3 / 4 proteins are also within the scope of protection of this application.

[0033] This invention utilizes hybridoma antibody technology to screen and obtain monoclonal antibodies against APOE, which are then used to construct immunochromatographic gold assay strips. These strips have been verified to simultaneously detect recombinant APOE2, APOE3, and APOE4 proteins, providing important reference value for the detection of APOE proteins and early warning of Alzheimer's disease or other abnormal risks.

[0034] Example 1

[0035] 1. Expression of recombinant proteins APOE2, APOE3, and APOE4

[0036] Based on the APOE4 amino acid sequence AAB59397.1, sequence analysis was performed, the N-terminal signal peptide (1-18 aa) was removed, and a start codon ATG was added to the N-terminus. The gene was synthesized by Qingke Biotechnology and cloned into the pET32a vector. Its nucleotide sequence is shown in SEQ ID NO.21:

[0037] AAAGTGGAACAGGCTGTTGAAACCGAACCAGAACCGGAACTTCGTCAGCAGACCGAATGGCAGTCTGGTCAGCGTTGGGAACTGGCTCTGGGTCGTTTCTGGGACTACCTGCGTTGGGTTCAGACCTTGTCTGAACAGGTTCAAGAAGAACTGCTGTCTTCTCAGGTTACTCAAGAACTGCGTGCGTTGATGGACGAAACCATGAAAGAACTGAAAGCGTACAAATCTGAACTGGAAGAACAGCTGACTCCAGTTGCAGAAGAAACTCGTGCTCGTCTGTCTAAAGAACTTCAGGCGGCTCAGGCGCGTCTGGGTGCTGACATGGAAGACGTTCGTGGTCGTCTGGTTCAGTACCGTGGTGAAGTTCAGGCAATGCTGGGTCAGTCTACCGAAGAACTGCGTGTTCGTCTGGCGAGCCATCTGCGTAAACTGCGTAAACGTCTGCTGCGTGATGCTGATGACCTTCAGAAACGTCTGGCTGTTTACCAGGCAGGTGCTCGTGAAGGTGCAGAACGTGGTCTGTCTGCTATCCGTGAACGTTTGGGTCCGCTGGTTGAACAGGGTCGTGTACGTGCAGCTACCGTTGGTTCTCTGGCAGGTCAGCCACTGCAAGAACGTGCTCAGGCATGGGGTGAACGTCTGCGTGCTCGTATGGAAGAAATGGGTTCTCGTACCCGTGATCGTCTGGACGAAGTGAAAGAACAGGTTGCTGAAGTTCGTGCGAAACTGGAAGAACAGGCGCAGCAGATCCGCCTCCAGGCGGAAGCGTTCCAGGCTCGTCTTAAGAGCTGGTTCGAGCCACTGGTTGAAGACATGCAGCGTCAGTGGGCAGGTCTGGTTGAGAAAGTTCAGGCTGCGGTTGGTACTTCCGCAGCTCCGGTTCCGTCTGACAACCAC。

[0038] The amino acid sequence is as shown in SEQ ID NO. 22:

[0039] KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVRGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLR DADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH.

[0040] APOE2 is created by site-directed mutation of APOE4's 112R and 158R to 112C and 158C, respectively, and then cloned into the pET32a vector.

[0041] The nucleotide sequence is as shown in SEQ ID NO.23: AAAGTGGAACAGGCTGTTGAAACCGAACCAGAACCGGAACTTCGTCAGCAGACCGAATGGCAGTCTGGTCAGCGTTGGGAACTGGCTCTGGGTCGTTTCTGGGACTACCTGCGTTGGGTTCAGACCTTGTCTGAACAGGTTCAAGAAGAACTGCTGTCTTCTCAGGTTACTCAAGAACTGCGTGCGTTGATGGACGAAACCATGAAAGAACTGAAAGCGTACAAATCTGAACTGGAAGAACAGCTGACTCCAGTTGCAGAAGAAACTCGTGCTCGTCTGTCTAAAGAACTTCAGGCGGCTCAGGCGCGTCTGGGTGCTGACATGGAAGACGTTTGCGGTCGTCTGGTTCAGTACCGTGGTGAAGTTCAGGCAATGCTGGGTCAGTCTACCGAAGAACTGCGTGTTCGTCTGGCGAGCCATCTGCGTAAACTGCGTAAACGTCTGCTGCGTGATGCTGATGACCTTCAGAAATGCCTGGCTGTTTACCAGGCAGGTGCTCGTGAAGGTGCAGAACGTGGTCTGTCTGCTATCCGTGAACGTTTGGGTCCGCTGGTTGAACAGGGTCGTGTACGTGCAGCTACCGTTGGTTCTCTGGCAGGTCAGCCACTGCAAGAACGTGCTCAGGCATGGGGTGAACGTCTGCGTGCTCGTATGGAAGAAATGGGTTCTCGTACCCGTGATCGTCTGGACGAAGTGAAAGAACAGGTTGCTGAAGTTCGTGCGAAACTGGAAGAACAGGCGCAGCAGATCCGCCTCCAGGCGGAAGCGTTCCAGGCTCGTCTTAAGAGCTGGTTCGAGCCACTGGTTGAAGACATGCAGCGTCAGTGGGCAGGTCTGGTTGAGAAAGTTCAGGCTGCGGTTGGTACTTCCGCAGCTCCGGTTCCGTCTGACAACCAC。

[0042] The amino acid sequence is as shown in SEQ ID NO.24:

[0043] KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLR DADDLQKCLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH.

[0044] APOE3 is created by site-directed mutation of 112R in APOE4 to 112C and then cloned into the pET32a vector.

[0045] The nucleotide sequence is as shown in SEQ ID NO.25: AAAGTGGAACAGGCTGTTGAAACCGAACCAGAACCGGAACTTCGTCAGCAGACCGAATGGCAGTCTGGTCAGCGTTGGGAACTGGCTCTGGGTCGTTTCTGGGACTACCTGCGTTGGGTTCAGACCTTGTCTGAACAGGTTCAAGAAGAACTGCTGTCTTCTCAGGTTACTCAAGAACTGCGTGCGTTGATGGACGAAACCATGAAAGAACTGAAAGCGTACAAATCTGAACTGGAAGAACAGCTGACTCCAGTTGCAGAAGAAACTCGTGCTCGTCTGTCTAAAGAACTTCAGGCGGCTCAGGCGCGTCTGGGTGCTGACATGGAAGACGTTTGCGGTCGTCTGGTTCAGTACCGTGGTGAAGTTCAGGCAATGCTGGGTCAGTCTACCGAAGAACTGCGTGTTCGTCTGGCGAGCCATCTGCGTAAACTGCGTAAACGTCTGCTGCGTGATGCTGATGACCTTCAGAAACGTCTGGCTGTTTACCAGGCAGGTGCTCGTGAAGGTGCAGAACGTGGTCTGTCTGCTATCCGTGAACGTTTGGGTCCGCTGGTTGAACAGGGTCGTGTACGTGCAGCTACCGTTGGTTCTCTGGCAGGTCAGCCACTGCAAGAACGTGCTCAGGCATGGGGTGAACGTCTGCGTGCTCGTATGGAAGAAATGGGTTCTCGTACCCGTGATCGTCTGGACGAAGTGAAAGAACAGGTTGCTGAAGTTCGTGCGAAACTGGAAGAACAGGCGCAGCAGATCCGCCTCCAGGCGGAAGCGTTCCAGGCTCGTCTTAAGAGCTGGTTCGAGCCACTGGTTGAAGACATGCAGCGTCAGTGGGCAGGTCTGGTTGAGAAAGTTCAGGCTGCGGTTGGTACTTCCGCAGCTCCGGTTCCGTCTGACAACCAC。

[0046] The amino acid sequence is as shown in SEQ ID NO.26:

[0047] KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLR DADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH.

[0048] Recombinant plasmids pET32a-APOE2, pET32a-APOE3, and pET32a-APOE4 were transformed into BL21(DE3) competent cells and induced to express their contents using standard methods. Specifically, the transformed bacteria were plated on LB agar plates (containing 100 μg / mL ampicillin) and incubated overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and incubated overnight at 37°C with shaking at 220 rpm. Finally, 1% of the total culture volume was inoculated into LB medium (containing 100 μg / mL ampicillin) and incubated at 37°C with shaking at 220 rpm for approximately 3 hours until OD500 reached. 600 The concentration was 0.6-0.9, and the final concentration was 0.1 mMIPTG. The cells were collected after induction at 30℃ and 200 rpm for 4 hours.

[0049] In the following text, when APOE2, APOE3 and APOE4 recombinant proteins are mentioned together, they will be collectively referred to as APOE2 / 3 / 4 recombinant proteins.

[0050] 2. Purification of APOE2 / 3 / 4 recombinant protein

[0051] Because the expressed recombinant protein carries a histidine tag, it was purified using a protein purification instrument and HisTrap from Suzhou Taidu Biotechnology Co., Ltd. TMPurification was performed using an HP affinity chromatography column. Buffer A consisted of 50 mM PB, 300 mM NaCl, pH 8.0; Buffer B consisted of 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The column was equilibrated with buffer A. The fermented bacterial culture was then centrifuged at 8000 rpm for 10 min. The precipitate was resuspended in buffer A and sonicated in ice water for 30 min, with 5-second intervals between sonications. The mixture was then centrifuged at 12000 rpm for 30 min. The supernatant was filtered through a 0.22 μm filter from JetBio and loaded onto the chromatography column. The column was washed with buffer A, followed by gradient elution with buffer B. The elution peak of the target protein was collected and dialyzed overnight at 4°C with buffer A. The purification process was observed by SDS-PAGE electrophoresis. The electrophoresis results of the purified protein are shown below. Figure 1 Protein concentration was determined using an ultra-micro spectrophotometer and stored at -20°C.

[0052] Figure 1 In the middle, M represents the protein marker, and numbers 1, 2, and 3 represent the purified APOE2 / 3 / 4 recombinant proteins, respectively. All three target protein bands are located around 55 kDa, which is consistent with the expected molecular weights (APOE2: 53.0 kDa, APOE3: 53.0 kDa, APOE4: 52.4 kDa), indicating successful expression and effective purification of the recombinant proteins. Each protein band is clear, single, and without obvious impurities, indicating high purity.

[0053] 3. Identification of APOE2 / 3 / 4 recombinant protein

[0054] Purified recombinant APOE2 / 3 / 4 proteins were coated onto microplates, and their reaction with APOE positive antibodies was identified by indirect ELISA. The APOE antibody was a commercially available APOE polyclonal antibody (Wuhan Sanying, 30535-1-AP). First, the recombinant proteins were coated into microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6), at a concentration of 1 μg / mL, 50 μL / well, incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 3% sucrose + 2% BSA, 150 μL per well, incubated at 37°C for 2 hours, followed by washing once with PBST wash buffer (PBS containing 0.05% Tween-20, pH 7.4), and then blotted dry. Polyclonal antibodies were diluted with PBS in gradients of 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL. 50 μL of each diluted antibody was added to each well of the antigen-coated microplate. A negative control was prepared by diluting purified rabbit IgG polyclonal antibody at the same concentration. The plates were incubated at 37°C for 30 min. The liquid in the wells was discarded, and the plates were washed four times with PBST. After drying, 50 μL / well of HRP-labeled goat anti-rabbit secondary antibody (Solepro, diluted 5000 times with PBS) was added. The plates were incubated at 37°C for 30 min, washed four more times, and dried. 50 μL / well of TMB chromogenic buffer was added, and the plates were incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meike Wande, 1001SA) was added to stop the reaction. The OD was measured using a microplate reader. 450 nm value. Results are as follows: Figure 2 The APOE2 / 3 / 4 antigens both bind to APOE polyclonal antibodies, and the purified APOE2 / 3 / 4 recombinant protein can be used for subsequent experiments.

[0055] 4. Screening of monoclonal antibodies that cross-react with APOE2 / 3 / 4 recombinant proteins

[0056] 4.1 Mouse Immunization

[0057] Since the three recombinant proteins APOE2, APOE3, and APOE4 differ only at a few amino acid sites, to obtain monoclonal antibodies more efficiently, mice were immunized with high-purity APOE3, and monoclonal antibody screening was performed using APOE2, APOE3, APOE4, and a reverse screening antigen. Specifically, purified APOE3 recombinant protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected at multiple sites into 6-week-old female BALB / c mice at a dose of 30 μg / mouse. In week 2, a booster immunization was performed by intramuscular injection of a mixture of 20 μg / mouse and an equal volume of MF59 adjuvant. In weeks 4 and 6, mice were immunized by direct injection of insulin into the spleen at a dose of 5 μg / mouse. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with high titers were selected for a booster immunization of 20 μg of APOE4 recombinant protein via intraperitoneal pulse, and the spleen was collected 3 days later for hybridoma cell preparation.

[0058] 4.2 Screening of hybridoma cells

[0059] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway to the bottom of the well, clones positive for APOE2, APOE3, and APOE4 recombinant proteins were selected by indirect ELISA. Since the immunogen was a prokaryotic expression source of the pET32a vector containing Trx and His tags, background components needed to be screened to identify specific cell lines targeting APOE protein. Positive cells were cloned to a monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0060] Indirect ELISA method for screening positive clones:

[0061] Recombinant proteins APOE2, APOE3, and APOE4, as well as other recombinant proteins of the pET32a vector (pET32a-HPV18 / E7, His tag, preparation method see patent 202510855254.X), were coated in microplates. The coating buffer was carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6. The coating concentration was 1 μg / mL, and the plate was incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 150 μL of 3% sucrose + 2% BSA per well, incubated at 37°C for 2 hours, and then washed once with PBST (PBS containing 0.05% Tween-20, pH 7.4). The plates were then blotted dry. 50 μL of cell culture supernatant was added, and the plate was incubated at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, pat dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, pat dry, and add 50 μL / well of TMB chromogenic buffer for incubation at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) to stop the reaction. Measure the OD using a microplate reader. 450 nm value. Positive cell lines that reacted with APOE2, APOE3, and APOE4 recombinant proteins but not with the control recombinant protein were selected for subsequent experiments.

[0062] Table 1. Screening results of monoclonal antibodies that cross-react with APOE2 / APOE3 / APOE4

[0063]

[0064] After the selected hybridoma cell lines were expanded and cultured, 0.2 ml (containing approximately 2.5 × 10⁻⁶ cells) was injected intraperitoneally into female BALB / c mice. 6 (1 cell), about 10 days later, when the mouse abdomen was significantly swollen, ascites fluid was collected using a sterile syringe needle.

[0065] 5. Purification of monoclonal antibodies that cross-react with APOE2 / APOE3 / APOE4

[0066] Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 ml of the supernatant, add 4 ml of acetate-sodium acetate buffer (0.06 M, pH 4.5), mix well, and slowly add 10 μl of n-octanoic acid while stirring. After the addition is complete, continue stirring for 30 minutes. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the supernatant. Filter the supernatant through defatted cotton, and add saturated ammonium sulfate at a final volume ratio of 50% (V / V) while stirring. After the addition is complete, continue stirring for 30 minutes, and let it precipitate overnight at 2–8°C. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the precipitate. After the precipitate was completely dissolved in binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), it was filtered through a 0.22 μm filter. The filtered sample was then pumped slowly through a peristaltic pump into a Protein L purification column equilibrated with binding buffer. The column was connected to a protein purification instrument, and the sample was washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled off. Elution was then performed with elution buffer (0.1 M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1 M Tris-HCl at pH 9 and placed in a dialysis bag (MW: 8000-14000). Dialysis was performed at 2-8 °C in 20 mM PBS pH 7.4 solution for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody.

[0067] Using the indirect ELISA method described above, the purified antibody was diluted to 1 μg / ml, and the reaction of the antibody with recombinant proteins APOE2, APOE3, and APOE4 was identified. Other recombinant proteins of the pET32a vector (pET32a-HPV18 / E7, His tag) were used as negative antigen controls. The results are shown in Table 2.

[0068] Table 2. Identification results of purified monoclonal antibodies that exhibit cross-reactivity with APOE2 / APOE3 / APOE4.

[0069]

[0070] 6. Preparation of test strips coated with different monoclonal antibodies against APOE:

[0071] The selected APOE monoclonal antibodies were scribed onto nitrocellulose membranes of different sizes (20 mm × 300 mm). Diluted monoclonal antibodies (diluted to 1.5 mg / mL with PBS at pH 7.4) were sprayed horizontally in a linear pattern using a scribing instrument, with a spray volume of 0.8 μL / cm per line, forming the detection line (T line). Goat anti-mouse IgG antibodies, diluted to 1 mg / mL with 0.01 M PBS at pH 7.4, were then sprayed horizontally in a linear pattern at 6 mm intervals, coating the nitrocellulose membrane at a volume of 0.8 μL / cm, forming the control line (C line).

[0072] 7. Colloidal gold pairing of APOE monoclonal antibodies

[0073] Preparation of antibody-colloidal gold labeled complex:

[0074] Antibody labeling: Colloidal gold solution was prepared using the trisodium citrate reduction method. The specific procedure was as follows: 100 mL of 0.01% chloroauric acid solution was heated to boiling, and then 1 mL of 1% trisodium citrate solution was quickly added until the solution turned wine-red. Boiling was continued for 5 minutes, and the colloidal gold particles were allowed to stabilize before cooling to room temperature. 1 mL of colloidal gold solution was placed in a centrifuge tube, and 0.2 M potassium carbonate solution was added in gradients of 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL to obtain the optimal pH for efficient antibody-colloidal gold conjugation. After mixing, 5 μg of the APOE monoclonal antibody to be labeled was added to each tube, and the mixture was quickly mixed and incubated at room temperature for 10 min. Then, 10 μL of 10% (w / v) bovine serum albumin (BSA) was added to block non-specific binding sites, and the mixture was incubated at room temperature for another 10 min. Add 10 μL of 10% (w / v) polyethylene glycol 20000 (PEG20000) to enhance labeling stability. After mixing, centrifuge at 12000 rpm for 10 min and discard the supernatant. Resuspend the lower precipitate in 1 / 10 volume of reconstitution solution (0.01 M phosphate buffer + 1% BSA + 2% sucrose, pH 7.4) to obtain the antibody-colloidal gold labeled complex. Store at 4°C protected from light for later use.

[0075] 8. Screening of paired monoclonal antibodies

[0076] Nitrocellulose membranes marked with different monoclonal antibodies against APOE were individually paired with different colloidal gold-labeled monoclonal antibodies. Since the amino acid differences between APOE2, APOE3, and APOE4 proteins are very small, one of the recombinant proteins was selected as a positive antigen for screening. Therefore, eukaryotically expressed APOE4 protein (Suzhou Nearshore, DRA288) was diluted to 20 ng / mL for detection, while HPV18 / E7 recombinant protein was diluted to 20 ng / mL as a negative antigen for detection. Combinations showing strong color development for APOE4 protein and not reacting with the control protein were selected. The results are shown in Table 3. Subsequently, the initial pairing was tested to verify the detection effect of the three proteins APOE2, APOE3, and APOE4. Prokaryotically expressed APOE2, APOE3, and APOE4 were diluted to 20 ng / mL for detection. Combinations showing strong color development for all three types of APOE protein were selected as the preferred pairings for further experiments. Both the control HPV18 / E7 recombinant protein and the blank dilution were negative. The results are shown in Table 4.

[0077] Table 3: Results of screening paired monoclonal antibodies using APOE recombinant protein

[0078]

[0079] Table 4: Results of screening paired monoclonal antibodies using APOE2 / 3 / 4 recombinant protein

[0080]

[0081] - indicates a negative result, meaning no color develops; + / ++ / +++ indicates a positive result, meaning a color reaction occurs. The more + signs there are, the deeper the color, and the stronger the positive reaction.

[0082] Twenty-seven monoclonal antibodies that reacted with all three types of APOE protein were streaked and labeled with gold, and then paired one-to-one. Table 3 shows that two pairings showed deeper color development: 3H7 streaking with 2C8 gold labeling; and 2C8 streaking with 4H3 gold labeling. These two combinations showed better detection of APOE4 protein. Furthermore, we compared the detection results of different types of recombinant APOE protein using three different types of APOE protein. As shown in Table 4, the 3H7 streaking and 2C8 gold labeling combination showed deep color development for all three types of recombinant APOE protein at 20 ng / mL, while the other combination showed weaker color development for some types. Therefore, the 3H7 streaking and 2C8 gold labeling combination was selected as the optimal pairing for detecting recombinant APOE protein.

[0083] 8. Preparation and assembly of colloidal gold test strips

[0084] Preparation of gold-labeled pads: A 6mm*300mm glass fiber membrane was treated with PBS containing 1% BSA and 1% Tween-20 at pH 7.4. The prepared colloidal gold-labeled antibody was then evenly added to the glass fiber membrane at a rate of 1200ul / strip. After air drying, the membrane was dried at 37℃ for 2 hours for later use.

[0085] See Figure 3 , Figure 3 This is a schematic diagram of the colloidal gold assembly. A 60mm*300mm PVC backing plate is used as a support, on which a sample pad, gold-labeled pad, nitrocellulose membrane, and absorbent paper are attached. The nitrocellulose membrane is coated with two lines and dried at 37℃ for 12 hours before use. The nitrocellulose membrane is coated with a detection line (monoclonal antibody 3H7 streaking) and a control line (goat anti-mouse IgG), and 2C8 gold is labeled. The assembled plate is cut into 4mm strips using a strip cutter and wrapped with a colloidal gold plastic casing, exposing the sample pad at the sample application well of the plastic casing, and exposing the control and detection lines at the result observation wells. The colloidal gold test strip is now assembled.

[0086] 9. Test strip specificity test

[0087] Recombinant protein samples: APOE2 recombinant protein, APOE3 recombinant protein, APOE4 recombinant protein, and HPV18 / E7 recombinant protein were diluted to 1 μg / mL with sample diluent for detection. 100 μL of the diluted sample was added to the sample well of the test strip. Simultaneously, 100 μL of diluent was added to a new test strip as a blank control. Results were determined within 20 minutes. A positive result was indicated by clear red bands on both the T and C lines; a negative result was indicated by only the C line; and an invalid result was indicated by no C line development.

[0088] Figure 4 The results showed that the test strip could effectively detect APOE2, APOE3, and APOE4 recombinant proteins, but showed no cross-reactivity with HPV18 / E7 recombinant proteins, indicating that the test strip has good specificity. (See attached image) Figure 4 BLK is the sample dilution solution: 0.01 MPB + 0.1% Tween 20 + 1.5% NaCl + 0.1% SDS, pH 7.4.

[0089] 10. Sensitivity test of test strips

[0090] The APOE2 recombinant protein, APOE3 recombinant protein, and APOE4 recombinant protein were diluted to the same concentration, then mixed in equal volumes to ensure that the concentrations of the three antigens in the mixed protein were the same. The mixture was then diluted to concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.25 ng / mL before detection. Figure 5The results showed that the colloidal gold test strip still showed weak color development at a recombinant protein concentration of 0.5 ng / mL, while the blank dilution, i.e. the sample dilution (0.01 MPB + 0.1% Tween 20 + 1.5% NaCl + 0.1% SDS, pH 7.4) (0 ng / mL), did not show color development, indicating that the limit of detection of the test strip for APOE recombinant protein is 0.5 ng / mL.

[0091] 11. Identification of the binding activity of paired antibodies to antigens

[0092] Referring to the aforementioned indirect ELISA method, the scratching antibody 3H7 and gold-labeled antibody 2C8 were serially diluted at 10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, and 100 pg / ml. Another murine-derived unrelated monoclonal antibody, HPV18 E7 monoclonal antibody (Santa Cruz, F-7), was used as a negative control (Ctrl). The binding activity of the antibodies to the APOE2 / 3 / 4 recombinant protein was measured. Results are as follows... Figure 6 The results showed that 3H7 and 2C8 still exhibited positive reactions with APOE2 / 3 / 4 at a dilution of 10 ng / ml, demonstrating high binding activity. Figure 6 The results, in order, show the binding reactions of recombinant proteins APOE2 (left), APOE3 (middle), and APOE4 (right) with the two antibodies.

[0093] 12. Variable region gene sequence of monoclonal antibodies

[0094] Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the light and heavy chains of the monoclonal antibody.

[0095] Sequence of membrane-scraped monoclonal antibody 3H7

[0096] The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 3H7 is shown in SEQ ID NO. 18:

[0097] GATATCCAGATGACACAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCACCTCTTGCAAGTCAAGTCAGAGCTCTTAGATAGTGGTGGAAAGACATATTTGAGTTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCCGGTGTCTACA CTGGACTCTGGAGTCCCTGACAGGTTCGCTGGCAGTGAATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGACTTTATTATTGCTGGCAAGGTACACATTTTCCCACGTGGGCGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTG.

[0098] Light chain amino acid sequence:

[0099] The amino acid sequence of the variable region of the light chain of monoclonal antibody 3H7 is shown in SEQ ID NO.14: DIQMTQTPLTLSVTIGQPASTSCKSSQSLLDSGGKTYLSWLLQRPGQSPKRLIYPVSTLDSGVPDRFAGSESGTDFTLKISRVEAEDLGLYYCWQGTHFPTWAFGGGTKLEIKRTV.

[0100] Light chain CDR area annotation:

[0101] The amino acid sequence CDR-L1 of the complementarity-determining region of the light chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO.4: CDR-L1: KSSQSLLDSGGKTYLS;

[0102] The amino acid sequence CDR-L2 of the complementarity-determining region of the light chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO. 5:

[0103] CDR-L2: PVSTLDS;

[0104] The amino acid sequence CDR-L3 of the complementarity-determining region of the light chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO. 6:

[0105] CDR-L3: WQGTHFPTWA.

[0106] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO.17:

[0107] CAGCGTGAGCTGCAGCAGTCTGGTGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCGGTAACTATGCCATGTCTTGGGTTCGCCAGACTCCCGAGAAGAGGCTGGAATGGGTCGCATCTATTGGTAATGGTGGTAGCGCCTTCTATCC AGACAGTGTGAAGGGCCGATTCACCATCTCCAGAAATAATGCCGGGAACACCCTGTTCCTGCAAATGAGCAGTCTGAGGTCTGAAGACACGGCCATGTATTATTGTACAAGAGGCGGGGACTATTATGATTACGACGGGACTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA.

[0108] Heavy chain amino acid sequence:

[0109] The amino acid sequence of the heavy chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO.13:

[0110] QRELQQSGGGLVKPGGSLKLSCAASGFTFGNYAMSWVRQTPEKRLEWVASIGNGSAFYPDSVKGRFTISRNNAGNTLFLQMSSLRSEDTAMYYCTRGGDYYDYDGTYWGQGTLVTVSS.

[0111] Heavy chain CDR region annotation:

[0112] The amino acid sequence of the complementarity-determining region (CDR-H1) of the heavy chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO. 1:

[0113] CDR-H1: NYAMS;

[0114] The amino acid sequence of the complementarity-determining region (CDR-H2) of the heavy chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO. 2:

[0115] CDR-H2: SIGNGGSAFYPDSVKG;

[0116] The amino acid sequence of the complementarity-determining region (CDR-H3) of the heavy chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO. 3:

[0117] CDR-H3: GGDYYDYDGTY.

[0118] The sequence of the gold monoclonal antibody 2C8:

[0119] Light chain nucleotide sequence:

[0120] The nucleotide sequence encoding the variable region of the 2C8 light chain of the monoclonal antibody is shown in SEQ ID NO.20:

[0121] GACATCCAGATGATTCAGTCTCCAGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGTAGGAGCCAGATGGCTGTTAGCACTAGCTACAGCAGCCACAGCTACTGGTATCAACAGAAATCAGGACAGCCACCCAAACTCCTCATCAAGAGCATGCTCTAC AGCGAGGCAGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATAGTGCAACATATTACTGTCCGCACCAGAGCCTGATCATCCCGTGGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGGTG.

[0122] The amino acid sequence of the variable region of the 2C8 light chain of the monoclonal antibody is shown in SEQ ID NO.16:

[0123] DIQMIQSPASLAVSLGQRATISCRSQMAVSTSYSSHSYWYQQKSGQPPKLLIKSMLYSEAGVPARFSGSGSGTDFTLNIHPVEEEDSATYYCPHQSLIIPWFGAGTKLELKRTV.

[0124] Light chain CDR area annotation:

[0125] The amino acid sequence of the CDR-L1 complementarity-determining region of the light chain of monoclonal antibody 2C8 is shown in SEQ ID NO.10:

[0126] CDR-L1: RSQMAVSTSYSSHSY;

[0127] The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain of monoclonal antibody 2C8 is shown in SEQ ID NO.11:

[0128] CDR-L2: SMLYSEA;

[0129] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain of monoclonal antibody 2C8 is shown in SEQ ID NO.12:

[0130] CDR-L3: PHQSLIIPW.

[0131] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2C8 is shown in SEQ ID NO.19: CAGCGTGAGCTGCAGCAGTCTGGACCTGGCCTGGTGGCGCCCTCACAGAGACTGTCCATCATTTGCACTGTCTCTGGATTTTCATTAACCAACTATGGTGTGCACTGGATTCGCCAGTCTCCAGGAAAGGGTCTAGAATGGCTGGGAGTTATTTGGGCTGGTGGAAATACAAATTATAATGCGGCTCTCATGTCCAGACTGGACATCAGTAAAGACAACTCCAAGAACCAAGTTTTCTTAGAAATGAACAGTCTGCAAACTGATGACACAGCCATTTATTATTGTGTCAGAGACGGTTATGACGTGGGGTTTGACCACTGGGGCCAAGGCACCACTCTCACTGTCTCTGCA.

[0132] The amino acid sequence of the heavy chain variable region of monoclonal antibody 2C8 is shown in SEQ ID NO.15:

[0133] QRELQQSGPGLVAPSQRLSIICTVSGFSLTNYGVHWIRQSPGKGLEWLGVIWAGGNTNYNAALMSRLDISKDNSKNQVFLEMNSLQTDDTAIYYCVRDGYDVGFDHWGQGTTLTVSA.

[0134] Heavy chain CDR region annotation:

[0135] The heavy chain variable region of monoclonal antibody 2C8 includes the amino acid sequence of the complementarity-determining region CDR-H1, as shown in SEQ ID NO. 7;

[0136] CDR-H1: NYGVH;

[0137] The heavy chain variable region of monoclonal antibody 2C8 includes the CDR-H2 amino acid sequence of the complementarity-determining region, as shown in SEQ ID NO. 8;

[0138] CDR-H2: VIWAGGNTNYNAALMS;

[0139] The heavy chain variable region of monoclonal antibody 2C8 includes the CDR-H3 amino acid sequence of the complementarity-determining region, as shown in SEQ ID NO. 9;

[0140] CDR-H3: DGYDVGFDH.

[0141] The embodiments of this application have now been described in detail. To avoid obscuring the concept of this application, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0142] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application.

Claims

1. A monoclonal antibody combination for simultaneous detection of APOE2 / 3 / 4 proteins, characterized in that, The monoclonal antibody combination includes monoclonal antibody 3H7 and monoclonal antibody 2C8. The heavy chain variable region of monoclonal antibody 3H7 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 3H7 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively. The heavy chain variable region of the monoclonal antibody 2C8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively. The light chain variable region of the monoclonal antibody 2C8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

2. The monoclonal antibody combination for simultaneous detection of APOE2 / 3 / 4 proteins according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for simultaneous detection of APOE2 / 3 / 4 proteins according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2C8 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 2C8 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for simultaneous detection of APOE2 / 3 / 4 proteins according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for simultaneous detection of APOE2 / 3 / 4 proteins according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2C8 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2C8 is shown in SEQ ID NO.

20.

6. The use of the monoclonal antibody combination according to claim 1 in the preparation of a monoclonal antibody tool for detecting APOE2 / 3 / 4 proteins.

7. The application according to claim 6, characterized in that, The tools include colloidal gold test strips, reagents, kits, and antibody chips.

8. The application according to claim 7, characterized in that, The colloidal gold test strip uses monoclonal antibody 3H7 as the capture antibody and monoclonal antibody 2C8 as the labeling antibody.

9. The application according to claim 8, characterized in that, The colloidal gold test strip includes a nitrocellulose membrane, a colloidal gold pad, a sample pad, and absorbent paper attached to a backing plate.

10. The application according to claim 9, characterized in that, The nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 3H7, the control line includes goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 2C8.

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