A kit, method and application for quantitatively detecting ApoE4 protein in blood

Abstract

The application discloses a kit, a method and application for quantitatively detecting ApoE4 protein in blood. The kit comprises magnetic beads coated with an ApoE capture antibody, an ApoE4 detection antibody labeled with a chemiluminescent label, and a pre-activation solution and an activation solution required for chemiluminescence detection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a kit, method, and application for the quantitative detection of ApoE4 protein in blood. Background Technology

[0002] In sporadic Alzheimer's disease (AD), apolipoproteins APOE Genes are the most significant risk factor, but the underlying mechanisms remain unclear. Apolipoprotein ApoE is mainly expressed in the brain and liver, participating in cholesterol and lipid transport in granulocytes. There are three isoforms of ApoE: ApoE ε2 (ApoE2), ApoE ε3 (ApoE3), and ApoE ε4 (ApoE4). All three isoforms have a strong affinity for Aβ oligomers and fibrinogen, thus becoming an important protein involved in the transport and clearance of β-amyloid (Aβ) in the body. The three isoforms and Aβ... 1-42 They all have similar high binding capacity. APOE ε4 heterozygotes and homozygotes have a higher risk of developing Alzheimer's disease than non-Alzheimer's disease. APOE The risk was 4.6 times and 14.9 times higher in ε4 carriers, respectively, and this risk increased further to 25.4 times in the elderly. APOE ε2 carriers offer some protection. Although these three isoforms differ by only two amino acids, their structures and functions are significantly different. These three isoforms and Aβ... 1-40 The binding abilities of ApoE4 and ApoE3 differ significantly, with ApoE2 exhibiting the highest binding capacity, followed by ApoE3, and then ApoE2 the lowest. This explains why... APOE The presence of ε4 as a high-risk factor for AD provides a clue. The varying binding affinity of ε4 to the low-density lipoprotein (LDL) receptor may be another important reason.

[0003] Testing individuals APOE Subtype analysis is very helpful in predicting AD risk. However, current methods require drawing peripheral blood, extracting DNA, and then using PCR for detection, which is time-consuming and expensive. More importantly, genetic testing cannot fully reflect the true expression of ApoE4 at the protein level. Developing a rapid detection kit to quickly determine the in vivo expression level of ApoE4 protein would be crucial for early AD screening, risk management, and early prevention. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art and provide a kit, method and application for quantitative detection of ApoE4 protein in blood.

[0005] To achieve the above objectives, the technical solution provided by this invention is as follows: The kit for quantitative detection of ApoE4 protein in blood includes magnetic beads coated with ApoE capture antibody, ApoE4 detection antibody labeled with chemiluminescent marker, and pre-excitation and excitation solutions required for chemiluminescent detection.

[0006] Preferably, the ApoE capture antibody is a mouse anti-human ApoE monoclonal antibody, the ApoE4 detection antibody is a rabbit anti-human ApoE4 antibody, and the chemiluminescent label is acridine ester.

[0007] More preferably, the kit further comprises sample diluent: 20 mM PB, 0.14 M NaCl, 5% BSA, 3% trehalose, 0.1% Brij-35, 0.05% PC300, 1% PEG20000 and 0.5% histidine, with a pH of 6.8.

[0008] More preferably, the kit further comprises an antibody diluent for diluting the chemiluminescently labeled ApoE4 detection antibody: 20 mM PB, 0.14 M NaCl, 2% BSA, 5% trehalose, 0.1% Brij-35, 0.05% PC300, 1% PEG20000, 1% histidine, and 1% mannitol, at a pH of 6.8.

[0009] More preferably, the kit further comprises a magnetic bead diluent for diluting the magnetic beads coated with ApoE capture antibody: 20 mM PB, 0.14 M NaCl, 1% BSA, 3% trehalose, 0.1% Brij-35, 0.05% PC300, 1% PEG20000 and 0.5% histidine, with a pH of 7.2-7.4.

[0010] The present invention also provides a method for detecting ApoE4 protein in blood samples based on the above-mentioned kit, the method comprising the following steps: (1) Mix the blood sample with the magnetic beads coated with ApoE capture antibody and perform a first incubation to allow the ApoE4 protein to bind to the capture antibody; (2) Perform magnetic separation and cleaning; (3) Add the ApoE4 detection antibody labeled with the chemiluminescent marker and perform a second incubation; (4) Perform magnetic separation and cleaning; (5) Add the pre-excitation solution and excitation solution in sequence, and detect the intensity of chemiluminescent photons.

[0011] Preferably, the magnetic beads coated with ApoE capture antibody are diluted at a ratio of 1:40 to 1:80.

[0012] Preferably, the blood sample is serum or plasma.

[0013] Preferably, the temperature for both the first and second incubation is 37°C, and the time for each is 5-10 minutes.

[0014] The kit described in this invention can be used to prepare in vitro diagnostic products for Alzheimer's disease risk screening.

[0015] The working principle of the reagent kit for detecting ApoE4 protein in blood of this invention is as follows: Picture 1 The process includes: A. Capturing antibodies covalently binding to magnetic beads; B. ApoE4 protein in the blood binding to antibody-coated magnetic beads; C. The ApoE4 protein captured by the magnetic beads binding to acridine ester (AE)-labeled antibodies. The concentration of ApoE4 protein can be quantified based on the intensity of acridine ester luminescence.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Direct detection of protein levels: Directly detects the ApoE4 protein content in the blood, which is more indicative of the actual protein expression in the body than gene testing.

[0017] 2. High specificity and sensitivity: It uses a specific detection antibody against the ApoE4 protein epitope, which can effectively distinguish ApoE4 from other ApoE subtypes, avoid cross-reaction, and has high detection sensitivity.

[0018] 3. Fast and efficient: The entire testing process takes only about 25 minutes and requires very little sample (5μL), making it ideal for rapid clinical screening.

[0019] 4. Accurate and reliable results: By optimizing the magnetic bead dilution ratio, incubation time, and adopting a two-step detection process, and by introducing an RF blocking agent, the accuracy and anti-interference ability of the detection are effectively improved.

[0020] 5. Clear application: This kit can be used to determine APOEε4 carrier status, providing a powerful tool for early risk warning and personalized health management of Alzheimer's disease.

[0021] In summary, apolipoprotein genes APOE The ε4 phenotype is the most significant known risk factor for sporadic Alzheimer's disease. This invention provides a kit for the rapid detection of ApoE ε4 (ApoE4) protein in blood using a CLIA immunochemiluminescence assay. This kit requires only approximately 5 µL of serum or plasma and can accurately quantify the ApoE4 protein content in a sample within 25 minutes, effectively distinguishing non-ApoE phenotypes from other types. APOE ε4 carriers APOE ε4 heterozygotes and APOE ε4 homozygote. Attached Figure Description

[0022] Picture 1 : Schematic diagram of the principle of immunochemiluminescence detection of ApoE4 protein.

[0023] Picture 2 ROC curves for distinguishing ε4 genotype carriers by ApoE4 protein concentration.

[0024] Picture 3 ROC curves for distinguishing homozygous and heterozygous ε4 genotypes by ApoE4 protein concentration. Detailed Implementation

[0025] Table 1 Solution preparation involved in specific embodiments

[0026] 1. Activation of magnetic beads 1) After vortexing the JSR carboxyl magnetic bead solution, take 5 mg of carboxyl magnetic bead solution into a 1.5 mL centrifuge tube, label it 1-2, magnetically aspirate for 1 min, and remove the supernatant. 2) Add 500 μL of 0.1M MES (pH 5.5) to the magnetic beads, vortex to disperse, magnetically adsorb for 1 min, remove the supernatant, and wash 3 times; 3) Add 500 μL of 0.1M MES (pH 5.5) solution, 50 µL of EDC solution (10 mg / mL), and 50 µL of NHS solution (10 mg / mL) to the magnetic beads, and vortex to disperse and mix well; (Note: EDC and NHS are prepared with 0.1M MES (pH 5.5) solution and must be prepared fresh for use.) 4) After vortexing and mixing the magnetic beads, place them on a vortex mixer and let them react at room temperature for 15 minutes; (Note: Vortex once every 10 minutes) 5) After the reaction is complete, the supernatant is removed by magnetic separation; 6) Add 500 μL of 0.1M MES (pH 5.5) to the magnetic beads, vortex to disperse, magnetically aspirate for 1 min, remove the supernatant, and wash twice.

[0027] 2. Antibody-magnetic bead conjugation 1) Add 500 μL of 0.1M MES (pH 5.5) solution containing 200 μg / mL mouse anti-human ApoE monoclonal antibody (Shenzhen Feipeng Biotechnology) to the magnetic beads, and vortex to mix (vortex gently to avoid generating bubbles); 2) Place the magnetic beads on a rotary instrument and react at room temperature for 3-4 hours; (Note: The magnetic beads are sonicated before antibody conjugation, sonication for 1 second, pause for 2 seconds, for a total of 3 minutes) 3) After the reaction is complete, the supernatant is removed by magnetic separation.

[0028] 3. Closure process 1) Add 1000 μL of small molecule blocking agent to the magnetic beads for blocking, place them on a rotary instrument and react at room temperature for 1 hour. After blocking, remove the supernatant by magnetic separation. 2) Add 1000 μL of washing buffer (TBS-T) to the magnetic beads, vortex disperse, magnetically separate to remove the supernatant, and wash 4 times; 3) Add 500 μL of magnetic bead preservation solution to the magnetic beads. (Note: After adding the preservation solution, the beads are ultrasonically broken up for 1 second, then paused for 2 seconds, for a total of 3 minutes.) 4. Labeling methods of acridine esters with different antibodies 1) 0.1 mg of ApoE4 antibody labeling; The stock solution concentration of rabbit anti-human ApoE4 antibody (Shenzhen Feipeng Biotechnology) was 7.15 mg / mL. Take 14 μL and add 386 μL of 50 mM NaHCO3 (pH 9.0) solution, with a total volume of 400 µL. The working solution concentration of anti-ApoE4 antibody was 0.25 mg / mL. 2) Preparation of acridine ester: Weigh 3 mg of acridine ester (NSP-SA-NHS) and dissolve it in 1000 μL of DMSO to obtain a stock solution concentration of 3 mg / mL. 3) Mix the protein and acridine ester at a molar ratio of 1:20. The antibody has a molecular weight of 150 kD, and the acridine ester (NSP-SA-NHS) has a molecular weight of 681.73. To label 0.1 mg of rabbit anti-human ApoE4 antibody, add 3.0 μL of acridine ester at a concentration of 3 mg / mL. 4) Place the container on a rotating apparatus and react at room temperature in the dark for approximately 0.5 hours; 5) Block with 1% glycine for 0.5 h; 6) Centrifuge the solution at 15000 rpm for 5 min; 7) Transfer the supernatant to a dialysis bag with a molecular weight of 50 kD for dialysis. Use 20 mM PB (pH 6.5-7.0) for dialysis of AE-ApoE4 at 400-450 rpm and 4℃. Use 1 L of dialysate each time, change the dialysate every 2-2.5 hours, change the dialysate 3 times in total, and perform dialysis 4 times. Perform dialysis overnight. 8) Collect the dialyzed solution, centrifuge at 15000 rpm for 5 min, and collect the supernatant; 9) Store at 4℃.

[0029] 5. Reagent preparation before use 1) Dilute the magnetic bead-ApoE antibody with diluent at a ratio of 1:40; The AE-ApoE4 antibody was diluted with diluent according to the specified ratio, and the luminescence value was controlled at around 20 million.

[0030] 2) Diluent: AE-ApoE4 antibody dilution buffer: 20mM PB, 0.14M NaCl, 2% BSA, 5% trehalose, 0.1% Brij-35, 0.05% PC300, 1% PEG20000, 1% histidine, 1% mannitol, pH 6.8; Magnetic bead-ApoE antibody dilution buffer: 20mM PB, 0.14M NaCl, 1% BSA, 3% trehalose, 0.1% Brij-35, 0.05% PC300, 1% PEG20000, 0.5% histidine, pH 7.2-7.4; Sample diluent: 20mM PB, 0.14M NaCl, 5% BSA, 3% trehalose, 0.1% Brij-35, 0.05% PC300, 1% PEG20000, 0.5% histidine, pH 6.8.

[0031] 6. The instrument operating procedure is as follows: Reaction system: 5 μL sample + 50 μL magnetic beads + 50 μL AE 1) Take a 5 μL sample; 2) Take 50 μL of magnetic beads; 3) Incubate at 37℃ for 10 minutes; 4) Cleaning; 5) Take 50 μL of acridinium ester-labeled antibody; 6) Incubate at 37℃ for 10 minutes; 7) Cleaning; 8) Add 100 μL of pre-activation solution (solution A) and 100 μL of activation solution (solution B) sequentially. 9) Detect the intensity of its chemiluminescent photons (detection wavelength 430nm).

[0032] 1. Testing the specificity of rabbit anti-human ApoE4 antibody This experiment used a combination of magnetic beads-ApoE antibody + AE-ApoE4 antibody to detect the standard. The experimental results are shown in Table 2. The results show that for the three antigens, the combination of magnetic beads-ApoE antibody + AE-ApoE4 antibody did not detect a significant reaction to ApoE2 and ApoE3 antigens, but a significant reaction was detected to ApoE4 antigen. Table 2. Magnetic bead-APOE antibody + AE-APOE4 antibody combination test for ApoE standards.

[0033] 2. Detect the ApoE4 protein content in old and fresh human blood samples. This experiment used a combination of magnetic bead-ApoE antibody and AE-ApoE4 antibody to detect fresh blood samples. The experimental results are shown in Tables 3 and 4. The results show that the combination of magnetic bead-ApoE antibody and AE-ApoE4 antibody can detect obvious reactions in both fresh serum and plasma samples.

[0034] Table 3. Magnetic bead ApoE antibody + AE-ApoE4 antibody combination test standard and blood samples after one week of storage.

[0035] Table 4. Detection of fresh plasma samples using magnetic bead-APOE antibody + AE-APOE4 antibody combination.

[0036] 3. Optimize Test 1 - Magnetic bead dilution ratio and incubation time This experiment used a combination of magnetic beads, APOE antibody, and AE-APOE4 antibody to detect the standard. The experimental results are shown in Tables 5 and 6. The results show that: 1) For different magnetic bead dilution ratios, the difference in luminescence values ​​between dilution ratios of 1:60 and 1:80 is not significant, and a dilution ratio of 1:80 can be used for subsequent tests; 2) For different incubation times, compared with 10 min + 5 min, shortening the reaction time increases the sensitivity, and an incubation time of 5 min + 5 min can be used for subsequent tests.

[0037] Table 5. Detection Standards with Different Magnetic Bead Dilution Ratios

[0038] Table 6. Standard samples for different incubation times

[0039] 4. Optimize Test 2 – Step-by-Step Method This experiment used three different reaction methods to test the combination of magnetic bead-ApoE antibody + AE-ApoE4 antibody. The experimental results are shown in Table 7. The results show that the two-step method has better luminescence value and luminescence intensity than the one-step method and the one-step delayed method. The two-step method can be used for subsequent tests.

[0040] Table 7. Detection of magnetic bead-ApoE antibody + AE-ApoE4 antibody using three reaction methods

[0041] Note: Reaction system: 5 μL sample + 50 μL magnetic beads + 50 μL AE; 1) One-step method: sample + magnetic beads + AE washing, incubation time 20 min; 2) One-step delayed method: sample + AE + magnetic beads washing, incubation time 10 min + 10 min; 3) Two-step method: sample + magnetic beads washing + AE, incubation time 10 min + 10 min.

[0042] 5. Optimization test 3 - The interference of rheumatoid factor (RF) on this test and the effect of eliminating this interference. This experiment used a combination of magnetic beads, ApoE antibody, and AE-ApoE4 antibody to test the interference of RF and verify the effect of the blocking agent. The experimental results are shown in Tables 8 and 9. The results show that: 1) For the standard with added RF, from the background level, the luminescence value increased with the increase of RF concentration, indicating the existence of false positive reactions. However, the luminescence value decreased after the addition of the blocking agent, indicating that the blocking agent has a certain inhibitory effect on false positives; 2) For serum and plasma samples, the luminescence value decreased after the addition of the blocking agent, indicating that the blocking agent can effectively reduce the interference level of RF in blood samples.

[0043] Table 8. Impact of RF Testing and Verification of the Effect of Blocking Agents

[0044] Table 9 Effects of Blockers on Blood Tests

[0045] 6. Determination of the limit of detection To determine the limit of detection (LOD) of our immunochemiluminescence method, we prepared a standard using a low concentration of ApoE4 protein (Shenzhen Feipeng Biotechnology). The results (Table 10) show that our method can detect ApoE4 protein as low as 50 ng / mL.

[0046] Table 10 Establishment of the standard curve

[0047] 7. Testing of clinical samples We randomly collected 143 EDTA-anticoagulated whole blood samples from Jing'an District Central Hospital in Shanghai and sent them to Hunan Sansure Biotech Co., Ltd. for genotyping. We also collected 42 known cases from Shanghai Mental Health Center. APOE To verify the reliability of our method, plasma samples were used for genotyping. APOE The genotyping results are only unblinded by the provider after quantitative results are obtained.

[0048] We first constructed a standard curve conforming to the human ApoE4 concentration range. Within the concentration range of 0-55 μg / mL, there was a significant curve correlation. After piecewise fitting with 4 PLCs, the correlation coefficient r > 0.99, and the deviation at each concentration point was within ±10% (Table 11). We then tested all clinical samples (Tables 12 and 13).

[0049] Table 11 Standard Curve Test Results

[0050] Table 12. Genotype and ApoE4 protein detection results of random samples from Jing'an District Central Hospital, Shanghai.

[0051] Table 13. Genotype and ApoE4 protein detection results from Shanghai Mental Health Center

[0052] The samples from the two hospitals were combined and statistically analyzed for each genotype and ApoE4 protein concentration, as follows: Table 14 Genotypes and ApoE4 Protein Concentration

[0053] ApoE4 protein concentration can distinguish ε4 genotype carrier status: based on genotyping results, among which... APOE Fifty-two individuals were carriers of the ε4 genotype, and 133 were non-carriers. ROC curve analysis was used to determine the differential diagnostic value of ApoE4 protein expression level for ε4 genotype carrier status. The results showed an AUC of 0.997, indicating that ApoE4 protein expression level has a very high diagnostic value for ε4 genotype carriers. Picture 2 Based on the maximum Youden index, the optimal cutoff value was determined to be 1.12. At this cutoff value, only one ε3 / ε3 carrier showed a false positive, with a diagnostic sensitivity of 1.000, specificity of 0.992, Youden index of 0.992, and accuracy of 99.5%. This indicates that using ApoE4 protein expression of 1.12 as the diagnostic threshold can effectively differentiate between ε3 / ε3 carriers. APOE ε4 genotype carriers and non-carriers.

[0054] ApoE4 protein concentration is insufficient to distinguish between ε4 heterozygotes and homozygotes: genotyping results are used as the standard. APOEFifteen cases were homozygous for the ε4 genotype, and 37 cases were heterozygous. ROC curve analysis showed an AUC of 0.514, close to the diagonal line, indicating poor discriminatory ability. The optimal threshold was 2.97 μg / mL, with a sensitivity of only 46.7%, specificity of 70.3%, and accuracy of 63.5%. Picture 3 Therefore, plasma ApoE4 protein concentration values ​​are difficult to effectively distinguish. APOE ε4 homozygotes and heterozygotes.

[0055] Conclusion: Our immunochemiluminescence immunoassay can effectively distinguish between different blood ApoE protein concentrations. APOE ε4 genotype carriers and non-carriers.

Claims

1. A kit for the quantitative detection of ApoE4 protein in blood, characterized in that, The kit includes magnetic beads coated with ApoE capture antibody, ApoE4 detection antibody labeled with chemiluminescent marker, and pre-excitation and excitation solutions required for chemiluminescent detection.

2. The kit for quantitative detection of ApoE4 protein in blood as described in claim 1, characterized in that, The ApoE capture antibody is a mouse anti-human ApoE monoclonal antibody, the ApoE4 detection antibody is a rabbit anti-human ApoE4 antibody, and the chemiluminescent label is acridine ester.

3. The kit for quantitative detection of ApoE4 protein in blood as described in claim 2, characterized in that, The kit also includes sample diluents: 20 mM PB, 0.14 M NaCl, 5% BSA, 3% trehalose, 0.1% Brij-35, 0.05% PC300, 1% PEG20000 and 0.5% histidine, with a pH of 6.

8.

4. The kit for quantitative detection of ApoE4 protein in blood as described in claim 3, characterized in that, The kit also includes an antibody diluent for diluting the chemiluminescently labeled ApoE4 detection antibody: 20 mMPB, 0.14 M NaCl, 2% BSA, 5% trehalose, 0.1% Brij-35, 0.05% PC300, 1% PEG20000, 1% histidine, and 1% mannitol, at pH 6.

8.

5. The kit for quantitative detection of ApoE4 protein in blood as described in claim 4, characterized in that, The kit also includes a magnetic bead diluent for diluting the magnetic beads coated with the ApoE capture antibody: 20 mM PB, 0.14 M NaCl, 1% BSA, 3% trehalose, 0.1% Brij-35, 0.05% PC300, 1% PEG20000 and 0.5% histidine, at a pH of 7.2–7.

4.

6. A method for detecting ApoE4 protein in a blood sample using the kit according to any one of claims 1 to 5, characterized in that, The method includes the following steps: (1) Mix the blood sample with the magnetic beads coated with ApoE capture antibody and perform a first incubation to allow the ApoE4 protein to bind to the capture antibody; (2) Perform magnetic separation and cleaning; (3) Add the ApoE4 detection antibody labeled with the chemiluminescent marker and perform a second incubation; (4) Perform magnetic separation and cleaning; (5) Add the pre-excitation solution and excitation solution in sequence, and detect the intensity of chemiluminescent photons.

7. The method for detecting ApoE4 protein in a blood sample based on the kit described in any one of claims 1 to 5, as described in claim 6, is characterized in that... The magnetic beads coated with ApoE capture antibody are diluted at a ratio of 1:40 to 1:

80.

8. The method for detecting ApoE4 protein in a blood sample based on the kit described in any one of claims 1 to 5, as described in claim 6, is characterized in that... The blood sample is serum or plasma.

9. The method for detecting ApoE4 protein in a blood sample based on the kit described in any one of claims 1 to 5, as described in claim 6, is characterized in that... The temperature for both the first and second incubation was 37°C, and the time for each was 5-10 minutes.

10. The use of the kit according to any one of claims 1 to 5 in the preparation of an in vitro diagnostic product for Alzheimer's disease risk screening.

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