Enzyme-linked immunosorbent assay method of apolipoprotein E and application of apolipoprotein E in diagnosis of glaucoma

Abstract

The invention belongs to the technical field of disease detection and health examination, and particularly relates to an enzyme-linked immunosorbent assay method of apolipoprotein E and application of the apolipoprotein E in glaucoma diagnosis. A capture antibody used by the kit is 1D4, a heavy chain variable region of the capture antibody comprises HCDR1-3 as shown in SEQ ID NO: 4-6, and a light chain variable region of the capture antibody comprises LCDR1-3 as shown in SEQ ID NO: 1-3; a detection antibody used by the kit is 3A6, a heavy chain variable region of the detection antibody comprises HCDR1-3 as shown in SEQ ID NO: 12-14, and a light chain variable region of the detection antibody comprises LCDR1-3 as shown in SEQ ID NO: 9-11. The double-antibody sandwich ELISA detection kit for detecting ApoE4 provided by the invention has no cross reaction with other subtypes of ApoEs, the used monoclonal antibody has high sensitivity, the kit can be used for screening glaucoma in physical examination and ophthalmic examination of old people, and the screening result is counted and summarized to a health information system and a cloud platform for big data analysis, so that the health condition of a patient can be detected.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay and health checkup technology, specifically relating to an enzyme-linked immunosorbent assay (ELISA) method for apolipoprotein E and its application in glaucoma diagnosis, which can be used for ophthalmological examinations in health checkup centers. Background Technology

[0002] Glaucoma, an irreversible blinding eye disease, has become one of the major visual health problems worldwide. Elevated intraocular pressure (IOP) is a common clinical feature of glaucoma. High IOP leads to damage to optic nerve fibers and apoptosis of retinal ganglion cells, ultimately manifesting as visual field defects and decreased vision; in severe cases, it can cause complete blindness. Treatments for glaucoma mainly include medication, laser therapy, and surgery. However, the primary goal of these treatments is to lower intraocular pressure and slow the progression of optic nerve damage; they cannot reverse existing optic nerve damage and vision loss. Moreover, even with well-controlled IOP, the disease may still progress. Glaucoma is insidious; it often presents with no obvious symptoms in its early stages, and patients often only discover it after significant vision loss, at which point optic nerve damage is often quite severe, missing the optimal intervention window.

[0003] Given the irreversibility and limitations of glaucoma treatment, early diagnosis and intervention are crucial for controlling the disease and protecting patients' visual function. In the early stages of the disease, optic nerve damage is mild, and visual field defects are often limited to minor changes in the peripheral field. At this time, effective intraocular pressure control and other treatments can significantly delay or even prevent the disease from progressing to the middle and late stages, maximizing the preservation of the patient's useful vision.

[0004] Although the causes of glaucoma are complex, its pathological mechanisms are all related to the apoptosis of retinal ganglion cells (RGCs) and Progressive optic disc remodeling is involved, and studies have shown that microglia, resident immune cells of the central nervous system (CNS), are key cell types leading to the loss of retinal ganglion cells (RGCs) in glaucoma. Furthermore, in the context of aging and neurodegenerative diseases, microglia shift from a homeostatic phenotype to an active phenotype, accompanied by the upregulation of pro-inflammatory cytokines, complement, and secretory factors such as apolipoprotein E (APOE). Further research suggests that APOE may serve as a potential biomarker for glaucoma, as detection has revealed significantly elevated levels of APOE in the aqueous humor of glaucoma patients compared to healthy individuals. Therefore, glaucoma can be diagnosed by detecting apolipoprotein E.

[0005] Based on this background, the present invention is dedicated to providing a detection technology for apolipoprotein E with high sensitivity and good specificity, in order to contribute to the prevention and treatment of glaucoma. Summary of the Invention

[0006] This invention provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting human apolipoprotein E. The capture antibody used in the kit is 1D4, whose heavy chain variable region includes HCDR1-3 as shown in SEQ ID NO: 4-6, and whose light chain variable region includes LCDR1-3 as shown in SEQ ID NO: 1-3. The detection antibody used in the kit is 3A6, whose heavy chain variable region includes HCDR1-3 as shown in SEQ ID NO: 12-14, and whose light chain variable region includes LCDR1-3 as shown in SEQ ID NO: 9-11.

[0007] Preferably, the heavy chain variable region of the 1D4 antibody is as shown in SEQ ID NO: 7.

[0008] Preferably, the light chain variable region of the 1D4 antibody is as shown in SEQ ID NO: 8.

[0009] Preferably, the heavy chain variable region of the 3A6 antibody is as shown in SEQ ID NO: 15.

[0010] Preferably, the light chain variable region of the 3A6 antibody is as shown in SEQ ID NO: 16.

[0011] Preferably, the kit further includes an enzyme-labeled plate, blocking solution, washing solution, and colorimetric solution.

[0012] Preferably, the detection antibody is labeled with horseradish peroxidase.

[0013] In a preferred embodiment of the present invention, the present invention provides an anti-ApoE4 monoclonal antibody composition comprising antibody 1D4, wherein the heavy chain variable region of the 1D4 antibody is shown as SEQ ID NO: 7, and the light chain variable region of the 1D4 antibody is shown as SEQ ID NO: 8.

[0014] Preferably, the anti-ApoE4 monoclonal antibody composition further includes antibody 3A6, wherein the heavy chain variable region of the 3A6 antibody is shown in SEQ ID NO: 15, and the light chain variable region of the 3A6 antibody is shown in SEQ ID NO: 16.

[0015] In another embodiment of the present invention, the present invention provides a method for using the anti-ApoE4 monoclonal antibody composition of the present invention for enzyme-linked immunosorbent assay (ELISA) detection of ApoE4, comprising the following steps: S1, use capture antibody-coated ELISA plate; S2, use sealing liquid for sealing; S3, discard the blocking solution, wash with buffer, then add the sample to be tested and incubate; S4, wash with buffer, then add detection antibody and incubate; S5, develop color and detect.

[0016] This invention provides a double-antibody sandwich ELISA kit for detecting ApoE4. When using this kit for double-antibody sandwich ELISA detection, it can specifically detect ApoE4 without cross-reactivity with other ApoE subtypes. The monoclonal antibody used can be prepared on a large scale and has high sensitivity. This invention can be used for glaucoma screening in ophthalmological examinations of the elderly, and the screening results can be statistically summarized in a health information system and cloud platform for big data analysis to monitor the patient's health status.

[0017] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0018] Figure 1 This is a scatter plot of mature ApoE4 protein detected by a double-antibody sandwich ELISA.

[0019] Figure 2 This is a graph showing the specific detection results of ApoE4 using a double-antibody sandwich ELISA. Detailed Implementation

[0020] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0021] Example 1: Preparation of Monoclonal Antibodies 1. Immunogen preparation ApoE4-specific monoclonal antibody immunogens were prepared by organic solid-phase synthesis, with amino acid sequences KLH-DMEDVRGRLVQYRGEV and KLH-RLLRDADDLQKRLAVY, respectively.

[0022] 2. Mouse immunization Two groups of SPF-grade Balb / c mice were selected and immunized with antigens KLH-DMEDVRGRLVQYRGEV and KLH-RLLRDADDLQKRLAVY, respectively. Each mouse was immunized with 50 μg of antigen per immunization. Before the first immunization, the immunogen was mixed with an equal volume of complete Freund's adjuvant. Thereafter, immunizations were performed every two weeks for a total of 3 immunizations. Blood was collected 10 days after the third immunization to separate serum and detect serum titers. Mice with high titers were selected and intraperitoneally injected again with 100 μg / 0.5 mL / mouse of antigen. Spleen cells were harvested 3 days later for fusion. Isolation of spleen cells and myeloma cells: Immunized mice were euthanized by cervical dislocation and disinfected by immersion in 75% alcohol; under aseptic conditions, the spleen was removed and placed in a culture dish, and the spleen tissue was minced with scissors; the spleen tissue was mixed with RPMI 1640 medium and filtered through a 70 μm cell sieve to prepare a spleen cell suspension; centrifuged at 1000 rpm for 10 minutes, the supernatant was discarded, and the cells were treated with erythrocyte lysis buffer to remove erythrocytes, followed by washing twice with RPMI 1640 medium; after cell counting, the cell concentration was adjusted to 1×10⁻⁶. 7 / mL for later use.

[0023] SP2 / 0 myeloma cells in logarithmic growth phase were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were then washed twice with RPMI 1640 medium, and the cell concentration was adjusted to 1×10⁻⁶ cells / mL. 7 / mL for later use.

[0024] Hybridoma cell fusion and screening: Spleen cells and myeloma cells were mixed at a ratio of 2:1 in a 50 mL centrifuge tube, centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded, ensuring no liquid residue remained in the tube. The bottom of the tube was gently tapped with a pipette to break up the cell clumps, and preheated 50% PEG 1450 solution was added slowly (over 60 seconds) while gently mixing. After PEG treatment for 90 seconds, preheated serum-free RPMI 1640 medium (5 mL) was quickly added to terminate the PEG treatment, and the mixture was allowed to stand for 10 minutes. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in HAT medium. The fused cell suspension was mixed with feeder cells (such as mouse peritoneal macrophages) and seeded into 96-well plates at 100 μL per well, ensuring that each well contained a single cell. The 96-well plates were incubated at 37°C in a 5% CO2 incubator, and cell growth was observed daily. The supernatant of positive clones was collected for detection.

[0025] Further testing was conducted on the affinity of monoclonal antibodies in the 33 positive clones, and the two strains with the best affinity were selected and named 1D4 and 3A6.

[0026] Further analysis was conducted to determine its affinity constants for ApoE2, ApoE3, and ApoE4. The results are shown in Table 1.

[0027] Table 1. Affinity constants of monoclonal antibodies

[0028] Table 2 provides the amino acid sequence of the complementarity-determining region (CDR) of the antibody.

[0029] Table 2

[0030] 1D4 VH variable heavy chain amino acid sequence QVELQQPGAELVRPQSSVKLSCKASQYTFT SNCGRTQ WVKQRPIQGLEWIG FSYDCTYLD KATLTVDKSSSTAYMQLSSLTSEDSAVYYCAR CYVPFSEDY WGQGTSVTVSS (SEQ ID NO: 7); 1D4 VL variable light chain amino acid sequence DIQMTQTTSSLSASLGDRVTISC SSVGDQ WYQQKPDGTIKLLIY SIKNTYPSGS GVPSRFSGSGSGTDYSLTISNLEPEDIATYYC QSGIVSMAPY FGTGTKLELK (SEQ ID NO: 8); 3A6 VH Variable Heavy Chain Amino Acid Sequence QVQLQQPGAELVKPGASVKLSCKASGYTFT LSQSA WVKQRPGQGLEWIG ASRVDYLPESQN KATLTVDKSSSTAYMQLSSLTSEDSAVYYCAR TYHHGS WGQGTTLTVSS (SEQ ID NO: 15); 3A6 VL Variable Light Chain Amino Acid Sequence DIVLTQSPASLAVSLGQRAIISC RQYTG WYQQKPGQQPKLLIY CDGGLDNGPVK GVPTRFSGSGSRTDFTLNIHPVEEDDAATYYC YQSNEHW FGGGTKLEIK (SEQ ID NO: 16).

[0031] Example 2: Detection of apolipoprotein E by double-antibody sandwich ELISA A double-antibody sandwich ELISA method was established for the detection of ApoE4. The selected antibody pairs were tested for detection sensitivity. 1D4 was used as the capture antibody and 3A6 was used as the detection antibody.

[0032] Experimental methods: 1. Coat the capture antibody 1D4, add 2 μg / mL of 1D4 to a 96-well plate, set two replicate wells, incubate overnight at 4°C, wash the plate 4 times with PBST the next day, and block with 5% skim milk powder at 37°C for 2 hours. 2. After blocking, wash the plate 4 times with PBST, add ApoE4 mature protein (concentration gradient of 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, 1.562 ng / mL, 0.781 ng / mL, 0 ng / mL) as primary antibody, 100 μL / well, and incubate at 37℃ for 1 hour; 3. After primary antibody incubation, wash the plate 4 times with PBST, add HRP-labeled 3A6 as secondary antibody (concentration of 2 μg / mL), and incubate at 37℃ for 1 hour; 4. After the secondary antibody incubation is complete, wash the plate 4 times with PBST, add 100 μL of single-component TMB colorimetric solution, develop the color at 37℃ for 10 minutes, add 50 μL of stop solution, and measure the absorbance at OD450-630nm to determine the binding activity.

[0033] like Figure 1 As shown, the relationship between the absorbance difference (OD450nm) and the concentration X of mature ApoE4 protein satisfies the equation Y = 0.7588x + 1.7436 (R² = 0.9949). It exhibits good linearity in the range of 1.562–50 ng / mL, with a detection limit as low as 0.781 ng / mL.

[0034] To investigate the specificity of the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for ApoE4, 1 ml of ApoE2, ApoE3, ApoE4 and PBS buffer were used as antigen samples for double-antibody sandwich assay.

[0035] The results are as follows Figure 2 As shown, the double-antibody sandwich detection method established by 1D4 and 3A6 of the present invention exhibits good specificity for ApoE4.

[0036] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. An enzyme-linked immunoassay kit for detecting human apolipoprotein E, wherein a capture antibody used in the kit is 1D4, the heavy chain variable region of which comprises HCDR1-3 as shown in SEQ ID NO: 4-6, and the light chain variable region of which comprises LCDR1-3 as shown in SEQ ID NO: 1-3; and a detection antibody used in the kit is 3A6, the heavy chain variable region of which comprises HCDR1-3 as shown in SEQ ID NO: 12-14, and the light chain variable region of which comprises LCDR1-3 as shown in SEQ ID NO: 9-11.

2. The double antibody sandwich assay kit according to claim 1, characterized in that, The heavy chain variable region of the 1D4 antibody is as shown in SEQ ID NO:

7.

3. The double antibody sandwich assay kit according to claim 1, characterized in that, The light chain variable region of the 1D4 antibody is as shown in SEQ ID NO:

8.

4. The double antibody sandwich assay kit of claim 1, wherein, The heavy chain variable region of the 3A6 antibody is as shown in SEQ ID NO:

15.

5. The double antibody sandwich assay kit of claim 1, wherein, The light chain variable region of the 3A6 antibody is as shown in SEQ ID NO:

16.

6. The double antibody sandwich assay kit of claim 1, wherein, The kit further comprises an enzyme-labeled plate, a blocking solution, a washing solution and a color developing solution.

7. The double antibody sandwich assay kit of claim 1, wherein, The detection antibody is labeled with horseradish peroxidase. 8.An anti-ApoE4 monoclonal antibody composition comprising antibody 1D4, the heavy chain variable region of which is as shown in SEQ ID NO: 7, and the light chain variable region of which is as shown in SEQ ID NO:

8.

9. The anti-ApoE4 monoclonal antibody composition of claim 8, wherein, The composition further comprises antibody 3A6, the heavy chain variable region of which is as shown in SEQ ID NO: 15, and the light chain variable region of which is as shown in SEQ ID NO:

16. 10.A method for using the anti-ApoE4 monoclonal antibody composition of claim 8 or 9 to perform enzyme-linked immunoassay of ApoE4, comprising the following steps: S1, coating an enzyme-labeled plate with a capture antibody; S2, blocking with a blocking solution; S3, discarding the blocking solution, washing with a buffer solution, then adding a sample to be detected, and incubating; S4, washing with a buffer solution, then adding a detection antibody, and incubating; S5, color developing and detecting.

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