Monoclonal antibody combination for simultaneously detecting APOE2 / 3 / 4 protein and application thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] 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. BACKGROUND

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

[0003] In humans, APOE is composed of 299 amino acids with a molecular weight of 34 kD, and has three major isoforms, APOE2, APOE3 and APOE4, which are encoded by APOE ɛ2, ɛ3 and ɛ4 alleles, respectively. The APOE gene is polymorphic in the human population, and the genotype of APOE also varies greatly among different populations. Variations in APOE alleles are believed to be associated with a variety of diseases such as atherosclerosis, nephrotic syndrome, diabetes and Alzheimer's disease. In addition to genetic polymorphism, plasma APOE protein levels can also reflect disease conditions. APOE2 has weak binding ability with low-density lipoprotein receptor (LDLR), which leads to its association with high plasma cholesterol levels and becomes a risk factor for atherosclerosis. APOE4 can significantly increase the total cholesterol concentration and plasma low-density lipoprotein level in healthy people, accelerate subjective cognitive decline and memory loss, and transform into mild cognitive impairment, and is considered a risk factor for cardiovascular disease and Alzheimer's disease. Research results show that the peripheral blood APOE protein level in patients with Alzheimer's disease is lower than that in healthy controls, and the decrease in plasma APOE protein level is positively correlated with the increased risk of Alzheimer's disease, and the risk of Alzheimer's disease-related dementia in the person with the lowest APOE protein level is still 3 times higher than that in the person with the highest APOE protein level, so lower APOE protein level is considered to be another risk factor independent of APOE ɛ4 gene. The "Expert Consensus on the Standardized Application of APOE ɛ4 Detection in Clinical Practice of Alzheimer's Disease" considers that low peripheral blood APOE protein level is independently associated with long-term risk of sporadic Alzheimer's disease, and recommends it as a biological marker for early screening of high-risk groups of Alzheimer's disease (class IIa recommendation, class B evidence). Low peripheral blood APOE protein level has potential value in predicting the progression of mild cognitive impairment (class IIb recommendation, class C evidence). Therefore, the detection of peripheral blood APOE protein has important significance for risk judgment and prediction of various diseases.

[0004] The current detection methods for plasma and serum APOE protein include radioimmunoassay and immunoturbidimetry. These methods have the problems of complex operation, long time consumption, strong equipment dependence, etc., and are difficult to meet the needs of rapid screening in primary medical institutions or on-site. Moreover, the existing commercial antibodies generally have the problems of insufficient recognition specificity for different isoforms of APOE, strong cross-reactivity, or insufficient sensitivity, etc., which makes it difficult to realize unified and accurate detection of APOE2, APOE3 and APOE4 proteins. Especially in the development of rapid detection tools (such as colloidal gold immunochromatographic test strips) based on the principle of double antibody sandwich, there is a lack of a pair of monoclonal antibodies with high affinity, high specificity and capable of simultaneously recognizing three isoforms, which seriously restricts the standardization and popularization of APOE protein detection technology. SUMMARY

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

[0006] In order to achieve the above-mentioned purposes, the main technical solutions adopted by the present application include: In a first aspect, the present application provides a monoclonal antibody combination for simultaneously detecting APOE2 / 3 / 4 proteins, which comprises monoclonal antibody 3H7 and monoclonal antibody 2C8, wherein the heavy chain variable region of the monoclonal antibody 3H7 comprises 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 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 4-SEQ ID NO. 6, respectively. The heavy chain variable region of the monoclonal antibody 2C8 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 7-SEQ ID NO. 9, respectively. The light chain variable region of the monoclonal antibody 2C8 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 10-SEQ ID NO. 12, respectively.

[0007] In further embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO. 14.

[0008] In a further embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2C8 is shown as SEQ ID NO. 15; the amino acid sequence of the light chain variable region of the monoclonal antibody 2C8 is shown as SEQ ID NO. 16.

[0009] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3H7 is shown as SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3H7 is shown as SEQ ID NO. 18.

[0010] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2C8 is shown as SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2C8 is shown as SEQ ID NO. 20.

[0011] In a second aspect, the application provides the use of the above-mentioned monoclonal antibody combination in the preparation of a monoclonal antibody tool for detecting APOE2 / 3 / 4 protein.

[0012] In a further embodiment, the tool comprises a colloidal gold detection test strip, a reagent, a kit and an antibody chip.

[0013] In a further embodiment, the colloidal gold detection test strip uses the monoclonal antibody 3H7 as a capture antibody and the monoclonal antibody 2C8 as a labeled antibody.

[0014] In a further embodiment, the colloidal gold detection test strip comprises a nitrocellulose membrane, a colloidal gold pad, a sample pad and an absorbent paper connected to a back plate.

[0015] In a further embodiment, the nitrocellulose membrane is provided with a detection line and a quality control line; the detection line is coated with the monoclonal antibody 3H7, the quality control line comprises goat anti-mouse IgG, and the colloidal gold pad is coated with the monoclonal antibody 2C8.

[0016] Advantages: The application provides a monoclonal antibody combination for simultaneously detecting APOE2 / 3 / 4 proteins, the combination comprising monoclonal antibody 3H7 and monoclonal antibody 2C8, which can simultaneously recognize three protein isomers APOE2, APOE3 and APOE4 with high specificity and high sensitivity, and is suitable for rapid detection reagent development. The monoclonal antibody 3H7 and the monoclonal antibody 2C8 have a clearly defined complementarity determining region (CDR) sequence, and the CDR amino acid sequences of the heavy chain and light chain variable regions are SEQ ID NO. 1-6 (3H7) and SEQ ID NO. 7-12 (2C8), respectively. The application further constructs a colloidal gold immunochromatographic test strip and other rapid detection tools. The results show that the detection sensitivity can reach 0.5 ng / mL, and there is no cross reaction with irrelevant proteins, which has extremely high specificity, provides a reliable tool for efficient and accurate detection of APOE protein, and is suitable for application in the fields of related scientific research and biological analysis, and also provides an efficient and reliable solution for clinical screening of APOE protein and risk assessment of related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative labor on the basis of these drawings.

[0018] Figure 1 The result graph of SDS-PAGE identification of proteins; Figure 2 The result graph of identification of APOE2 (A), APOE3 (B) and APOE4 (C) recombinant proteins; Figure 3 The schematic diagram of colloidal gold assembly; Figure 4 The specificity analysis result graph of the test strip; Figure 5 The sensitivity test result graph of the test strip; Figure 6 The binding reaction of APOE2 (left), APOE3 (middle) and APOE4 (right) recombinant proteins with monoclonal antibodies. DETAILED DESCRIPTION

[0019] The embodiments of the present application will be further described in detail below in combination with the drawings and examples. The detailed description of the following examples and the drawings are used to exemplarily illustrate the principles of the present application, but cannot be used to limit the scope of the present application, and the present application can be implemented in many different forms, not limited to the specific embodiments disclosed in the text, but includes all technical solutions falling within the scope of the claims.

[0020] The present application provides these examples is to make the present application thorough and complete, and to the person skilled in the art fully express the scope of the present application. It should be noted that: unless otherwise specified, the relative arrangement of components and steps, the components of the material, the numerical expression and the numerical values set forth in these examples should be interpreted as merely exemplary, and not as a limitation.

[0021] APOE2 / 3 / 4 protein includes APOE2 / 3 / 4 recombinant protein and natural protein.

[0022] Using the monoclonal antibody combination 3H7 and 2C8 protected by the present application, detecting APOE2, APOE3, APOE4, APOE2 / 3 or APOE2 / 4, APOE3 / 4 protein alone also belongs to the protection scope of the present application.

[0023] The present application obtains APOE monoclonal antibody by hybridoma antibody technology, and constitutes an immunogold chromatography test paper, which is verified to be able to simultaneously detect APOE2, APOE3, APOE4 recombinant protein, and has important reference significance in the detection of APOE protein, early warning of Alzheimer's disease or other abnormal risks.

[0024] Example 1 1. APOE2, APOE3 and APOE4 recombinant protein expression According to the APOE4 amino acid sequence AAB59397.1, sequence analysis is carried out, the N-terminal signal peptide (1-18 aa) is removed, and the start codon ATG is added at the N-terminal. The gene is synthesized by GenScript and cloned into pET32a vector, and the nucleotide sequence is shown as SEQ ID NO. 21: AAAGTGGAACAGGCTGTTGAAACCGAACCAGAACCGGAACTTCGTCAGCAGACCGAATGGCAGTCTGGTCAGCGTTGGGAACTGGCTCTGGGTCGTTTCTGGGACTACCTGCGTTGGGTTCAGACCTTGTCTGAACAGGTTCAAGAAGAACTGCTGTCTTCTCAGGTTACTCAAGAACTGCGTGCGTTGATGGACGAAACCATGAAAGAACTGAAAGCGTACAAATCTGAACTGGAAGAACAGCTGACTCCAGTTGCAGAAGAAACTCGTGCTCGTCTGTCTAAAGAACTTCAGGCGGCTCAGGCGCGTCTGGGTGCTGACATGGAAGACGTTCGTGGTCGTCTGGTTCAGTACCGTGGTGAAGTTCAGGCAATGCTGGGTCAGTCTACCGAAGAACTGCGTGTTCGTCTGGCGAGCCATCTGCGTAAACTGCGTAAACGTCTGCTGCGTGATGCTGATGACCTTCAGAAACGTCTGGCTGTTTACCAGGCAGGTGCTCGTGAAGGTGCAGAACGTGGTCTGTCTGCTATCCGTGAACGTTTGGGTCCGCTGGTTGAACAGGGTCGTGTACGTGCAGCTACCGTTGGTTCTCTGGCAGGTCAGCCACTGCAAGAACGTGCTCAGGCATGGGGTGAACGTCTGCGTGCTCGTATGGAAGAAATGGGTTCTCGTACCCGTGATCGTCTGGACGAAGTGAAAGAACAGGTTGCTGAAGTTCGTGCGAAACTGGAAGAACAGGCGCAGCAGATCCGCCTCCAGGCGGAAGCGTTCCAGGCTCGTCTTAAGAGCTGGTTCGAGCCACTGGTTGAAGACATGCAGCGTCAGTGGGCAGGTCTGGTTGAGAAAGTTCAGGCTGCGGTTGGTACTTCCGCAGCTCCGGTTCCGTCTGACAACCAC.

[0025] The amino acid sequence is shown in SEQ ID NO. 22: KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVRGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH.

[0026] APOE2 is that 112R, 158R of APOE4 are respectively site-mutated into 112C, 158C, and cloned into pET32a vector; The nucleotide sequence is set forth in SEQ ID NO. 23: AAAGTGGAACAGGCTGTTGAAACCGAACCAGAACCGGAACTTCGTCAGCAGACCGAATGGCAGTCTGGTCAGCGTTGGGAACTGGCTCTGGGTCGTTTCTGGGACTACCTGCGTTGGGTTCAGACCTTGTCTGAACAGGTTCAAGAAGAACTGCTGTCTTCTCAGGTTACTCAAGAACTGCGTGCGTTGATGGACGAAACCATGAAAGAACTGAAAGCGTACAAATCTGAACTGGAAGAACAGCTGACTCCAGTTGCAGAAGAAACTCGTGCTCGTCTGTCTAAAGAACTTCAGGCGGCTCAGGCGCGTCTGGGTGCTGACATGGAAGACGTTTGCGGTCGTCTGGTTCAGTACCGTGGTGAAGTTCAGGCAATGCTGGGTCAGTCTACCGAAGAACTGCGTGTTCGTCTGGCGAGCCATCTGCGTAAACTGCGTAAACGTCTGCTGCGTGATGCTGATGACCTTCAGAAATGCCTGGCTGTTTACCAGGCAGGTGCTCGTGAAGGTGCAGAACGTGGTCTGTCTGCTATCCGTGAACGTTTGGGTCCGCTGGTTGAACAGGGTCGTGTACGTGCAGCTACCGTTGGTTCTCTGGCAGGTCAGCCACTGCAAGAACGTGCTCAGGCATGGGGTGAACGTCTGCGTGCTCGTATGGAAGAAATGGGTTCTCGTACCCGTGATCGTCTGGACGAAGTGAAAGAACAGGTTGCTGAAGTTCGTGCGAAACTGGAAGAACAGGCGCAGCAGATCCGCCTCCAGGCGGAAGCGTTCCAGGCTCGTCTTAAGAGCTGGTTCGAGCCACTGGTTGAAGACATGCAGCGTCAGTGGGCAGGTCTGGTTGAGAAAGTTCAGGCTGCGGTTGGTACTTCCGCAGCTCCGGTTCCGTCTGACAACCAC.

[0027] The amino acid sequence is set forth in SEQ ID NO. 24: KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKCLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH.

[0028] APOE3 is site-directed mutation of 112R of APOE4 to 112C and cloned into pET32a vector; The nucleotide sequence is set forth in SEQ ID NO. 25: AAAGTGGAACAGGCTGTTGAAACCGAACCAGAACCGGAACTTCGTCAGCAGACCGAATGGCAGTCTGGTCAGCGTTGGGAACTGGCTCTGGGTCGTTTCTGGGACTACCTGCGTTGGGTTCAGACCTTGTCTGAACAGGTTCAAGAAGAACTGCTGTCTTCTCAGGTTACTCAAGAACTGCGTGCGTTGATGGACGAAACCATGAAAGAACTGAAAGCGTACAAATCTGAACTGGAAGAACAGCTGACTCCAGTTGCAGAAGAAACTCGTGCTCGTCTGTCTAAAGAACTTCAGGCGGCTCAGGCGCGTCTGGGTGCTGACATGGAAGACGTTTGCGGTCGTCTGGTTCAGTACCGTGGTGAAGTTCAGGCAATGCTGGGTCAGTCTACCGAAGAACTGCGTGTTCGTCTGGCGAGCCATCTGCGTAAACTGCGTAAACGTCTGCTGCGTGATGCTGATGACCTTCAGAAACGTCTGGCTGTTTACCAGGCAGGTGCTCGTGAAGGTGCAGAACGTGGTCTGTCTGCTATCCGTGAACGTTTGGGTCCGCTGGTTGAACAGGGTCGTGTACGTGCAGCTACCGTTGGTTCTCTGGCAGGTCAGCCACTGCAAGAACGTGCTCAGGCATGGGGTGAACGTCTGCGTGCTCGTATGGAAGAAATGGGTTCTCGTACCCGTGATCGTCTGGACGAAGTGAAAGAACAGGTTGCTGAAGTTCGTGCGAAACTGGAAGAACAGGCGCAGCAGATCCGCCTCCAGGCGGAAGCGTTCCAGGCTCGTCTTAAGAGCTGGTTCGAGCCACTGGTTGAAGACATGCAGCGTCAGTGGGCAGGTCTGGTTGAGAAAGTTCAGGCTGCGGTTGGTACTTCCGCAGCTCCGGTTCCGTCTGACAACCAC.

[0029] The amino acid sequence is set forth in SEQ ID NO. 26: KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH.

[0030] The recombinant plasmids pET32a-APOE2, pET32a-APOE3, pET32a-APOE4 were transformed into BL21 (DE3) competent cells respectively and induced to express according to the conventional method. Specifically, the transformed bacteria were spread on LB agar plates (containing 100 μg / mL ampicillin) and incubated at 37°C overnight. Single colonies were picked and inoculated into 5 mL LB medium (containing 100 μg / mL ampicillin) and incubated at 37°C, 220 rpm overnight. The culture was inoculated into LB medium (containing 100 μg / mL ampicillin) at a volume of 1% of the total volume of the medium, and incubated at 37°C, 220 rpm for about 3 hours until the OD 600 was 0.6-0.9. IPTG was added at a final concentration of 0.1 mM, and the bacteria were induced at 30°C, 200 rpm for 4 hours, and then collected.

[0031] Hereinafter, when referring to APOE2, APOE3 and APOE4 recombinant proteins simultaneously, they are collectively referred to as APOE2 / 3 / 4 recombinant proteins.

[0032] 2. Purification of APOE2 / 3 / 4 recombinant proteins Since the expressed recombinant proteins have a histidine tag, the protein purification instrument, HisTrap TMHP affinity chromatography column for purification. A buffer is 50 mM PB, 300 mM NaCl, pH 8.0, B buffer is 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0, the chromatography column with A buffer equilibration, then the fermentation bacteria 8000 rpm centrifugation 10 min, the precipitate with A liquid resuspended, in ice water ultrasonic broken 30 min, interval 5 seconds ultrasonic 5 seconds, 12000 rpm centrifugation 30 min, the supernatant with the Jiete biology 0.22 micron filter filter after the chromatography column, the chromatography column with A buffer washing, followed by B buffer gradient elution. The purpose of protein elution peak with A buffer dialysis at 4 ℃ overnight, the purified protein by SDS-PAGE electrophoresis observation. The purified protein electrophoresis results as shown in Figure 1 The protein concentration was determined by ultramicro spectrophotometer and stored at -20 ℃.

[0033] Figure 1 M: protein marker, the serial numbers 1, 2, 3 are: purified APOE2 / 3 / 4 recombinant protein. Three target protein bands are located near 55 kDa, which is basically consistent with the expected molecular weight (APOE2: 53.0 kDa, APOE3: 53.0 kDa, APOE4: 52.4 kDa), indicating that the recombinant protein is successfully expressed and effectively purified, each protein band is clear, single, no obvious band, high purity.

[0034] 3, identification of APOE2 / 3 / 4 recombinant protein The purified recombinant APOE2 / 3 / 4 proteins were coated on the enzyme-labeled plate respectively, and the indirect ELISA method was used to identify the reaction with APOE positive antibody, which was a commercial APOE polyclonal antibody (Wuhan Three Yings, 30535-1-AP). First, the recombinant protein was coated in the microplate (coating buffer: carbonate buffer, sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, constant volume to 1 L of pure water, pH 9.6), the coating concentration was 1 μg / mL, 50 μL / well, 4°C overnight. The next day, the coating solution was discarded, and 3% sucrose + 2% BSA was used for blocking, 150 μL per well, 37°C for 2 hours, then washed with PBST (0.05% Tween-20 in PBS, pH 7.4) once, and dried. The polyclonal antibody was diluted with PBS at a gradient of 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / ml, and 50 μL was added to the antigen-coated microplate, and the purified rabbit IgG polyclonal antibody was diluted at the same concentration as the negative control, 37°C for 30 min. The liquid in the well was shaken out, and the plate was washed with PBST 4 times, and then dried. 50 μL / well of HRP-labeled goat anti-rabbit secondary antibody (Solebao, diluted 5000 times with PBS) was added, 37°C for 30 min, and then washed 4 times. After drying, 50 μL / well of TMB color developing liquid was added, and color development was performed at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) was added to terminate the reaction, and the OD 450 nm value. The results are shown in Figure 2 . APOE2 / 3 / 4 antigens have binding reactions with APOE polyclonal antibodies, and the purified APOE2 / 3 / 4 recombinant proteins can be used for subsequent experiments.

[0035] 4. Screening of monoclonal antibodies cross-reacted with APOE2 / 3 / 4 recombinant proteins 4.1, Mouse immunization Since the three types of APOE2, APOE3, and APOE4 recombinant proteins only have individual amino acid site differences, in order to obtain monoclonal antibodies more efficiently, the APOE3 recombinant protein with higher purity was selected to immunize mice, and APOE2, APOE3, APOE4 and counter-screening antigens were used for monoclonal antibody screening. Specifically, the purified APOE3 recombinant protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected into 6-week-old female BALB / c mice at a dose of 30 μg per mouse. At week 2, the mice were boosted with 20 μg per mouse mixed with an equal volume of MF59 adjuvant by intramuscular injection. At weeks 4 and 6, the mice were immunized by directly injecting the spleen with 5 μg per mouse. Seven days after the last immunization, the mouse serum was tested for antibody titer. The mouse with higher titer was selected for intraperitoneal boost with 20 μg of APOE4 recombinant protein, and the mouse spleen was taken 3 days later for preparation of hybridoma cells.

[0036] 4.2 Screening of hybridoma cells 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.

[0037] Indirect ELISA method for screening positive clones: 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.

[0038] Table 1. Screening results of monoclonal antibodies that cross-react with APOE2 / APOE3 / APOE4

[0039] 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. 6The ascites was collected with a sterile syringe needle about 10 days later when the abdomen of the mouse was obviously swollen.

[0040] 5. Purification of the APOE2 / APOE3 / APOE4 cross-reactive monoclonal antibody The ascites was centrifuged at 12000 r / min for 10 minutes, 1 ml of supernatant was taken, 4 ml of acetic acid-sodium acetate buffer (0.06M, pH 4.5) was added, mixed evenly, then 10 μl of n-octanoic acid was slowly added while stirring, after adding, continue to stir for 30 minutes, centrifuge at 12000 r / min for 30 minutes at 2-8°C, take the supernatant. The supernatant was filtered with defatted cotton, and saturated ammonium sulfate was added at a ratio of 50% (V / V) of the final volume, and stirred while adding. After adding, continue to stir for 30 minutes, precipitate overnight at 2-8°C, centrifuge at 12000 r / min for 30 minutes at 2-8°C, take the precipitate. After the precipitate was completely dissolved with binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), it was filtered through a 0.22 μm filter, and the filtered sample was pumped into a Protein L purification column equilibrated with binding buffer at low speed through a peristaltic pump. Connect the protein purification instrument, wash with 5-10 column volumes of binding buffer until the ultraviolet absorption peak is washed flat, then elute with elution buffer (0.1M glycine, pH 2.7), collect the elution peak, and adjust the collected sample to neutral with 1M Tris-HCl, pH 9, load into a dialysis bag (MW: 8000-14000), dialyze in 20 mM PBS pH 7.4 solution at 2-8°C for 16 hours. Transfer the liquid in the dialysis bag to a centrifuge tube and centrifuge at 12000 r / min for 5 minutes. The supernatant is the purified monoclonal antibody.

[0041] Using the above indirect ELISA method, the purified antibody was diluted to 1 ug / ml, and the reaction of the antibody with APOE2, APOE3, and APOE4 recombinant proteins was identified. Other recombinant proteins of pET32a vector (pET32a-HPV18 / E7, His tag) were used as negative antigen controls. The results are shown in Table 2.

[0042] Table 2. Identification results of purified APOE2 / APOE3 / APOE4 cross-reactive monoclonal antibody

[0043] 6. Preparation of test strips coated with different APOE monoclonal antibodies The selected APOE monoclonal antibodies were respectively drawn on the nitrocellulose membrane with different specifications of 20 mm x 300 mm, using a membrane drawing instrument to spray the diluted monoclonal antibodies (diluted to 1.5 mg / mL with PBS with pH 7.4) in a horizontal line, with a spraying amount of 0.8 uL / cm, to form a test line (T line). With an interval of 6 mm, the goat anti-mouse IgG antibody was sprayed in a horizontal line with the same spraying amount, with a dilution concentration of 1 mg / mL in 0.01 M PBS with pH 7.4, coated on the nitrocellulose membrane with an amount of 0.8 uL / cm, to form a control line (C line).

[0044] 7. Gold colloid pairing of APOE monoclonal antibody Preparation of antibody-gold colloid labeling complex: Antibody labeling: The gold colloid solution was prepared by the trisodium citrate reduction method. Specifically, 100 mL of 0.01% chloroauric acid solution was heated and boiled, then 1 mL of 1% trisodium citrate solution was quickly added until the solution color was wine red. Boiling was continued for 5 minutes, and after the gold colloid particles were stable, the solution was cooled to room temperature for standby. 1 mL of gold colloid solution was taken into a centrifuge tube, and 0.2 M potassium carbonate solution was added in a gradient of 1 uL, 2 uL, 3 uL, 4 uL, 5 uL, and 6 uL to obtain the pH for efficient coupling of the antibody and gold colloid. After mixing, 5 ug of the APOE monoclonal antibody to be labeled was added, and after rapid mixing, it was incubated at room temperature for 10 minutes. Then, 10 uL of 10% (w / v) bovine serum albumin (BSA) was added to block the non-specific binding sites, and incubation was continued at room temperature for 10 minutes. Then, 10 uL of 10% (w / v) polyethylene glycol 20000 (PEG20000) was added to enhance the labeling stability, and after mixing, it was centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded. The lower precipitate was resuspended with 1 / 10 volume of resuspension solution (0.01 M phosphate buffer + 1% BSA + 2% sucrose, pH 7.4), to obtain the antibody-gold colloid labeling complex, which was stored at 4°C in the dark for standby.

[0045] 8. Screening of paired monoclonal antibodies The nitrocellulose membrane with different monoclonal antibodies of APOE drawn was respectively paired with the different monoclonal antibodies labeled with colloidal gold. Because the amino acid difference of APOE2, APOE3 and APOE4 protein is very small, one of the recombinant proteins was selected as a positive antigen for screening. Therefore, the eukaryotic expressed APOE4 protein (Suzhou Coast, DRA288) was diluted to 20 ng / mL for detection, and the HPV18 / E7 recombinant protein was diluted to 20 ng / mL as a negative antigen for detection, and the combination with deep color reaction to APOE4 protein and no reaction to the control protein was screened. The results are shown in Table 3. Subsequently, the detection effect of the initial screening pair on three proteins APOE2, APOE3 and APOE4 was tested, and the prokaryotic expressed APOE2, APOE3 and APOE4 were diluted to 20 ng / mL for detection, and the combination with strong color reaction to the three types of APOE protein was selected as the preferred pair for the next test. The control HPV18 / E7 recombinant protein and blank diluent were negative. The results are shown in Table 4.

[0046] Table 3: Results of screening paired monoclonal antibodies with APOE recombinant protein

[0047] Table 4: Results of screening paired monoclonal antibodies with APOE2 / 3 / 4 recombinant protein

[0048] - represents negative, i.e. no color; + / ++ / +++ represents positive, i.e. color reaction, the more the number of +, the deeper the color, i.e. the stronger the positive reaction.

[0049] The 27 strains of monoclonal antibodies reacting with three types of APOE protein were respectively drawn and labeled with gold, and were paired one by one. The results of Table 3 showed that the color of two paired combinations was deep, which were 3H7 drawn membrane, 2C8 labeled gold; 2C8 drawn membrane, 4H3 labeled gold. The above two combinations had good detection effect on APOE4 protein. Further, we compared the detection results of different types of APOE recombinant protein with three different types of APOE protein. As shown in Table 4, the combination of 3H7 drawn membrane and 2C8 labeled gold had deep color reaction to 20 ng / mL of three types of APOE recombinant protein, while the other combination had weak color reaction to part of the types, so the combination of 3H7 drawn membrane and 2C8 labeled gold was selected as the best pair for detecting APOE recombinant protein.

[0050] 8. Preparation and assembly of colloidal gold detection test paper Preparation of gold mark pad: 6mm*300mm glass fiber membrane was treated with PBS containing 1% BSA and 1% Tween-20, pH 7.4, and then the prepared colloidal gold labeled antibody was uniformly added to the glass fiber membrane at 1200ul / strip. After natural drying, it was placed at 37℃ for 2h for standby; See Figure 3 , Figure 3 The schematic diagram of colloidal gold assembly is shown in Figure 1. A 60mm*300mm PVC backboard was used as support, and sample pad, gold mark pad, nitrocellulose membrane and water absorption paper were attached to it respectively. Two lines were coated on the nitrocellulose membrane, which was dried at 37℃ for 12h for standby. The nitrocellulose membrane was coated with detection line (monoclonal antibody 3H7) and quality control line (goat anti-mouse IgG). The assembled large plate was cut into 4mm naked strips with a cutting machine, and wrapped with colloidal gold plastic shell. The sample pad was exposed to the sample hole position of the plastic card shell, and the quality control line and detection line were exposed to the result observation hole position. The colloidal gold test paper card assembly was completed.

[0051] 9. Specificity test of test strip Recombinant protein samples: APOE2 recombinant protein, APOE3 recombinant protein, APOE4 recombinant protein, and HPV18 / E7 recombinant protein were diluted to 1ug / mL with sample diluent for detection. 100uL of the above diluted samples were added to the sample hole of the test paper card, and 100uL of the diluent was added to a new test paper card as a blank control. The results were determined within 20min. If T line and C line appeared clear red bands, it was judged as positive; only C line developed color was negative; if C line did not develop color, it was judged as invalid.

[0052] Figure 4 The results showed that the test paper card could well detect APOE2 recombinant protein, APOE3 recombinant protein, and APOE4 recombinant protein, and had no cross reaction with HPV18 / E7 recombinant protein, indicating that the test paper card had good specificity. Figure 4 BLK is sample diluent, 0.01M PB+0.1% Tween20+1.5% NaCl+0.1% SDS, pH 7.4.

[0053] 10. Sensitivity test of test strip APOE2 recombinant protein, APOE3 recombinant protein, and APOE4 recombinant protein were diluted to the same concentration, then mixed in equal volume, so that the concentration of the three antigens in the mixed protein was the same, and then diluted to 100ng / mL, 10ng / mL, 1ng / mL, 0.5ng / mL, and 0.25ng / mL for detection. Figure 5The results show that the colloidal gold test paper still has weak coloration at a recombinant protein concentration of 0.5 ng / mL, while the blank diluent, i.e., the sample diluent (0.01 M PB + 0.1% Tween 20 + 1.5% NaCl + 0.1% SDS, pH 7.4) (0 ng / mL) does not color, indicating that the minimum detection limit of the test paper card for APOE recombinant protein is 0.5 ng / mL.

[0054] 11. Identification of the binding activity of the paired antibody to the antigen According to the foregoing indirect ELISA method, the membrane antibody 3H7 and the labeled antibody 2C8 were gradient diluted at 10 ug / ml, 1 ug / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, 100 pg / ml, and other irrelevant mouse-derived single antibodies: HPV18 E7 monoclonal antibody (Santa Cruz, F-7) were used as negative controls (Ctrl) to determine the binding activity of the antibodies to APOE 2 / 3 / 4 recombinant proteins. The results are shown in Figure 6 The results show that 3H7 and 2C8 still have positive reactions with APOE 2 / 3 / 4 at a dilution of 10 ng / ml, and have high binding activity. Figure 6 The results are, in order, the binding reactions of APOE 2 (left), APOE 3 (middle), and APOE 4 (right) recombinant proteins with the 2 antibodies.

[0055] 12. Sequences of the variable region genes of the monoclonal antibodies Total RNA of the hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using Random Primers; mouse antibody variable region universal primers were designed, and the VH and VL genes were amplified by 2 rounds of PCR, and an Age1 and Bsiw1 enzyme cutting site was introduced into the primer in the 3rd round of PCR. The PCR product was cut and purified, and then ligated to the pUC19 vector, and the TOP10 strain was transformed and cultured at 37°C for 14 h, and then single colonies were picked for sequencing to obtain the gene sequences of the light and heavy chains of the monoclonal antibodies.

[0056] Sequence of the membrane monoclonal antibody 3H7 The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO. 18: GATATCCAGATGACACAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCACCTCTTGCAAGTCAAGTCAGAGCTCTTAGATAGTGGTGGAAAGACATATTTGAGTTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCCGGTGTCTACA CTGGACTCTGGAGTCCCTGACAGGTTCGCTGGCAGTGAATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGACTTTATTATTGCTGGCAAGGTACACATTTTCCCACGTGGGCGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTG.

[0057] Light chain amino acid sequence: The amino acid sequence of the variable region of the light chain of monoclonal antibody 3H7 is shown in SEQ ID NO.14: DIQMTQTPLTLSVTIGQPASTSCKSSQSLLDSGGKTYLSWLLQRPGQSPKRLIYPVSTLDSGVPDRFAGSESGTDFTLKISRVEAEDLGLYYCWQGTHFPTWAFGGGTKLEIKRTV.

[0058] Light chain CDR area annotation: 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; 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: CDR-L2: PVSTLDS; 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: CDR-L3: WQGTHFPTWA.

[0059] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO.17: CAGCGTGAGCTGCAGCAGTCTGGTGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCGGTAACTATGCCATGTCTTGGGTTCGCCAGACTCCCGAGAAGAGGCTGGAATGGGTCGCATCTATTGGTAATGGTGGTAGCGCCTTCTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAAATAATGCCGGGAACACCCTGTTCCTGCAAATGAGCAGTCTGAGGTCTGAAGACACGGCCATGTATTATTGTACAAGAGGCGGGGACTATTATGATTACGACGGGACTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA.

[0060] Heavy chain amino acid sequence: The amino acid sequence of the heavy chain variable region of monoclonal antibody 3H7 is shown as SEQ ID NO. 13: QRELQQSGGGLVKPGGSLKLSCAASGFTFGNYAMSWVRQTPEKRLEWVASIGNGGSAFYPDSVKGRFTISRNNAGNTLFLQMSSLRSEDTAMYYCTRGGDYYDYDGTYWGQGTLVTVSS.

[0061] Heavy chain CDR region annotation: The amino acid sequence of the complementarity determining region CDR-H1 of the heavy chain variable region of monoclonal antibody 3H7 is shown as SEQ ID NO. 1, respectively: CDR-H1: NYAMS; The amino acid sequence of the complementarity determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 3H7 is shown as SEQ ID NO. 2, respectively: CDR-H2: SIGNGGSAFYPDSVKG; The amino acid sequence of the complementarity determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 3H7 is shown as SEQ ID NO. 3, respectively: CDR-H3: GGDYYDYDGTY.

[0062] Monoclonal antibody 2C8 sequence: Light chain nucleotide sequence: The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2C8 is shown as SEQ ID NO. 20: GACATCCAGATGATTCAGTCTCCAGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGTAGGAGCCAGATGGCTGTTAGCACTAGCTACAGCAGCCACAGCTACTGGTATCAACAGAAATCAGGACAGCCACCCAAACTCCTCATCAAGAGCATGCTCTACAGCGAGGCAGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATAGTGCAACATATTACTGTCCGCACCAGAGCCTGATCATCCCGTGGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGGTG.

[0063] The amino acid sequence of the light chain variable region of the monoclonal antibody 2C8 is shown as SEQ ID NO. 16: DIQMIQSPASLAVSLGQRATISCRSQMAVSTSYSSHSYWYQQKSGQPPKLLIKSMLYSEAGVPARFSGSGSGTDFTLNIHPVEEEDSATYYCPHQSLIIPWFGAGTKLELKRTV.

[0064] The light chain CDR region is labeled as: The amino acid sequence of the light chain complementarity determining region CDR-L1 of the monoclonal antibody 2C8 is shown as SEQ ID NO. 10, respectively: CDR-L1: RSQMAVSTSYSSHSY; The amino acid sequence of the light chain complementarity determining region CDR-L2 of the monoclonal antibody 2C8 is shown as SEQ ID NO. 11, respectively: CDR-L2: SMLYSEA; The amino acid sequence of the light chain complementarity determining region CDR-L3 of the monoclonal antibody 2C8 is shown as SEQ ID NO. 12, respectively: CDR-L3: PHQSLIIPW.

[0065] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2C8 is shown as SEQ ID NO. 19: CAGCGTGAGCTGCAGCAGTCTGGACCTGGCCTGGTGGCGCCCTCACAGAGACTGTCCATCATTTGCACTGTCTCTGGATTTTCATTAACCAACTATGGTGTGCACTGGATTCGCCAGTCTCCAGGAAAGGGTCTAGAATGGCTGGGAGTTATTTGGGCTGGTGGAAATACAAATTATAATGCGGCTCTCATGTCCAGACTGGACATCAGTAAAGACAACTCCAAGAACCAAGTTTTCTTAGAAATGAACAGTCTGCAAACTGATGACACAGCCATTTATTATTGTGTCAGAGACGGTTATGACGTGGGGTTTGACCACTGGGGCCAAGGCACCACTCTCACTGTCTCTGCA.

[0066] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2C8 is shown as SEQ ID NO. 15: QRELQQSGPGLVAPSQRLSIICTVSGFSLTNYGVHWIRQSPGKGLEWLGVIWAGGNTNYNAALMSRLDISKDNSKNQVFLEMNSLQTDDTAIYYCVRDGYDVGFDHWGQGTTLTVSA.

[0067] Heavy chain CDR region annotation: The heavy chain variable region of the monoclonal antibody 2C8 includes the amino acid sequence of the complementarity determining region CDR-H1 shown as SEQ ID NO. 7; CDR-H1: NYGVH; The heavy chain variable region of the monoclonal antibody 2C8 includes the amino acid sequence of the complementarity determining region CDR-H2 shown as SEQ ID NO. 8; CDR-H2: VIWAGGNTNYNAALMS; The heavy chain variable region of the monoclonal antibody 2C8 includes the amino acid sequence of the complementarity determining region CDR-H3 shown as SEQ ID NO. 9; CDR-H3: DGYDVGFDH.

[0068] So far, the embodiments of the present application have been described in detail. In order to avoid obscuring the concept of the present application, some details known in the art are not described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein according to the above description.

[0069] Although some specific embodiments of the present application have been described in detail through examples, those skilled in the art should understand that the above examples are only for illustration, not for limiting the scope of the present application. Those skilled in the art should understand that the above embodiments can be modified or some technical features can be replaced equivalently without departing from the scope and spirit of the present application.

Claims

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

2. The monoclonal antibody combination for simultaneously detecting APOE 2 / 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 simultaneously detecting APOE 2 / 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 simultaneously detecting APOE 2 / 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 simultaneously detecting APOE 2 / 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. Use of the monoclonal antibody combination according to claim 1 in the preparation of a monoclonal antibody tool for detecting APOE2 / 3 / 4 protein.

7. Use according to claim 6, characterized in that, The tool comprises colloidal gold detection test strips, reagents, kits and antibody chips.

8. Use according to claim 7, characterized in that, The colloidal gold detection test strip takes monoclonal antibody 3H7 as a capture antibody and monoclonal antibody 2C8 as a labeled antibody.

9. Use according to claim 8, characterized in that, The colloidal gold detection test strip comprises a nitrocellulose membrane connected to a back plate, a colloidal gold pad, a sample pad and an absorbent paper.

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

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