Preparation of ABEI macrolide and application of ABEI macrolide in preparation of AD diagnostic kit

Abstract

The invention discloses preparation of ABEI macrolide and application of ABEI macrolide in preparation of an AD diagnostic kit, ABEI macrolide is adopted as a marker, the chemiluminescence reaction does not need to depend on horse radish peroxidase (HRP) and a traditional enhancer, and a detection system is simpler. The AD diagnostic kit provided by the invention comprises a reagent 1 containing magnetic beads labeled by a p-Tau181 antibody, a reagent 2 containing a p-Tau181 antibody labeled by ABEI macrolide, a p-Tau181 antigen standard substance and excitation liquid, and the provided AD diagnostic kit has the advantages of high stability, simplicity and convenience in operation, light drifting resistance, high sensitivity and low cost, and can provide more accurate detection guarantee for clinical diagnosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of immunodetection, and particularly relates to a preparation of ABEI macrolide and application of the ABEI macrolide in an AD diagnostic kit for detecting content of p-Tau181 protein by using a chemiluminescence method. BACKGROUND

[0002] The prevalence of Alzheimer's disease (AD) increases exponentially with age: about 3% for 65-74 years old, about 10% for 75-84 years old, and up to 30% for 85 years old and above. According to the data of the National Bureau of Statistics, there were about 308 million people aged 60 and above in China in 2023, and it is expected to exceed 400 million in 2050. Therefore, in the diagnosis of Alzheimer's disease, we need to continuously optimize and innovate on the basis of existing diagnostic reagents and detection technologies to make more accurate diagnoses for clinical practice.

[0003] The pathological process of AD begins with the deposition of amyloid beta (Aβ), followed by the over-phosphorylation of tau protein and the formation of neurofibrillary tangles, ultimately leading to neuronal death and cognitive decline. p-Tau181 is the product of tau protein phosphorylation at threonine 181, and its level increases in synchrony with the occurrence of tau pathology, which is several years or even decades earlier than clinical symptoms such as memory loss. Normal elderly people may experience mild cognitive decline with age, but the level of p-Tau181 is usually normal; even if the cognitive symptoms of early AD patients are mild, the level of p-Tau181 has already increased significantly, which can be used as a marker of "pathological aging". As a specific biomarker of AD, p-Tau181 can not only identify AD pathology and distinguish different types of dementia in the preclinical stage, but also monitor disease progression and aid drug development. Currently, the diagnostic reagents for diagnosing AD biomarkers by chemiluminescence on the market are mostly direct light-emitting reagents labeled with acridinium ester or indirect light-emitting reagents labeled with luminol. The existing diagnostic reagents have the following defects: the acridinium ester label has a short shelf life (usually only one month) and poor light bleaching resistance. The luminol label requires the addition of horseradish peroxidase and enhancer, and the reagent stability is poor. SUMMARY

[0004] The present application provides a preparation method of ABEI macrolide and its application in the preparation of an AD diagnostic kit for detecting the content of p-Tau181 protein by chemiluminescence, which contains a p-Tau181 antibody labeled with ABEI macrolide. The diagnostic reagent has the advantages of strong stability, simple operation, light bleaching resistance, high sensitivity, and low cost, and can provide more accurate detection for clinical diagnosis.

[0005] To achieve the purpose of the application, the present application provides the following technical solutions: One, the application also provides a preparation method of ABEI macrolide, specifically comprising the following steps: S1. N-(4-aminobutyl)-N-ethyl isorumin is added to anhydrous pyridine solvent in a molar ratio of 1:1.2-1.5, and the reaction is stirred at 50℃ for 20 min. After the reaction is completed, the ABEI-glutarate intermediate is obtained by column chromatography purification; S2. The prepared ABEI-glutarate intermediate is added to a mixed solvent of DMF / acetonitrile=1:1 v / v in a molar ratio of 1.2-2.0:1, and the reaction is reacted at 60℃ for 120 h. After the reaction is completed, the ABEI macrolide is obtained by column chromatography purification. The structural formula of the ABEI macrolide is shown in the accompanying Figure 5 .

[0006] The ABEI macrolide provided by the preparation method has a stable macrocyclic structure formed by C atoms and a hydroxyl functional group. The macrocyclic structure stability and π conjugation enhance the light emission, solve the steric hindrance problem of luminol and the pH sensitivity defect of acridinium ester, and have high labeling rate and low detection limit, which provides a basis for the development of chemical luminescent reagents with more stability, light bleaching resistance and high sensitivity.

[0007] Since there is no commercially available product of ABEI macrolide at present, the synthesis method of ABEI macrolide provided by the application optimizes the synthesis from the aspects of improving the yield, shortening the reaction time, simplifying the purification steps and enhancing the stability of the product, which significantly improves the purity and yield of the target product ABEI macrolide. The purity can reach 92%, and the yield is increased to 56%.

[0008] Two, the application also provides a method for labeling p-Tau181 antibody with ABEI macrolide, comprising the following steps: (1) The p-Tau181 antibody is subjected to dialysis purification treatment to sufficiently remove residual ammonium ions and other small molecule interferents in the sample; (2) ABEI macrolide and p-Tau181 antibody are coupled: the molar ratio of ABEI macrolide to p-Tau181 antibody is 20:1; The specific procedures for the coupling reaction are as follows: ABEI macrolide was washed twice by centrifugation using 0.05M MES buffer, pH 5.0; 200 μL of MES buffer, 3.5 μL of 10 mg / mL EDC, and 14 μL of 10 mg / mL NHS were added, and the carboxyl group was activated in an ice-water bath for 20 minutes; after centrifugation, it was redispersed in 0.05M MES buffer, pH 5.0; 0.1 mL of 10 μg / mL p-Tau181 antibody was added, mixed, and reacted at 0°C in the dark for 2 hours; 0.02 mL of 1% BSA was added to block the carboxyl activation site for 20-40 minutes, and unreacted p-Tau181 antibody was removed by centrifugation. The mixture was then redispersed in 0.05M MES buffer and stored at 4°C for later use.

[0009] In step (2) above, the process of labeling p-Tau181 antibody with ABEI macrolide is achieved by linking the carboxyl group of ABEI macrolide and the amino group of p-Tau181 antibody through an amide chemical bond.

[0010] This application uses ABEI macrolide as a marker, and its chemiluminescent reaction does not rely on horseradish peroxidase (HRP) and traditional enhancers (such as p-iodophenol and luminol enhancers), making the detection system simpler and achieving beneficial technical effects.

[0011] III. This invention provides the application of ABEI macrolide in the preparation of an AD diagnostic kit for the chemiluminescence detection of p-Tau181 protein content.

[0012] This invention provides an AD diagnostic kit for detecting p-Tau181 protein content using chemiluminescence immunoassay, characterized by comprising the following reagents: Reagent 1: Magnetic beads labeled with p-Tau18 antibody; Reagent 2 contains p-Tau181 antibody labeled with ABEI macrolide; p-Tau181 antigen standard and activation solution.

[0013] Reagent 1 comprises magnetic beads labeled with p-Tau18 antibody, and PBS buffer containing bovine serum albumin (BSA) and sodium azide with a pH of 7.4; Reagent 2 includes an ABEI macrolide-labeled p-Tau181 antibody and a buffer containing Tris-HCl pH=8.0 and ProClin 300. The p-Tau181 antigen standard includes 11 standard samples with p-Tau181 protein concentrations of 0 pg / mL, 0.05 pg / mL, 0.1 pg / mL, 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL and 500 pg / mL.

[0014] The p-Tau181 antigen standard is prepared by diluting recombinant p-Tau181 protein with a purity of ≥95% in a PBS buffer containing 5% fetal bovine serum, 0.05% Tween-20, 0.01% ProClin 300 and having a pH value of 7.4.

[0015] The p-Tau181 antigen standard is used to construct a standard curve, calibrate the quantitative accuracy of the kit through the correspondence between the known concentration of the antigen and the luminescence signal, and ensure the reliability of the sample detection results.

[0016] The excitation solution contains Tris-HCl and H2O2, preferably Tris-HCl with a pH of 8.5 and a concentration of 100 mM, and H2O2 with a content of 0.1-0.5% (v / v).

[0017] Tris-HCl maintains the alkaline environment of the reagent and promotes the dissociation of ABEI into a luminescent active form. H2O2 is used to trigger the oxidative luminescence reaction of ABEI.

[0018] The function of the excitation solution is that ABEI reacts with H2O2 under alkaline conditions (pH maintained by Tris-HCl) to undergo oxidative dehydrogenation and release photons. Reaction formula: ABEI + H2O2 → oxidized ABEI* + 2H2O, where ABEI* is in an excited state and releases photons when it returns to the ground state.

[0019] The method for detecting the content of p-Tau181 protein in a sample using the AD diagnostic kit provided by the present application is as follows: (1) Add the detection sample, and prepare the reaction system with reagent 1 and reagent 2: the p-Tau18 antibody-labeled magnetic beads contained in reagent 1 specifically capture the p-Tau181 antigen in the sample, and the ABEI macrolide-labeled p-Tau181 antibody contained in reagent 2 binds to the p-Tau181 antigen captured by the magnetic beads to form a “magnetic bead-antibody-p-Tau181-antibody-ABEI” complex; (2) Detect the luminescence intensity: after the reaction is completed and the magnetic separation and washing are completed, the excitation solution is added, ABEI releases a chemiluminescence signal, the luminescence intensity is detected, and the luminescence intensity is positively correlated with the concentration of the p-Tau181 antigen, so as to obtain the content of the p-Tau181 protein in the sample.

[0020] The p-Tau18 antibody labeled magnetic beads contained in the reagent 1 and the ABEI macrolide labeled p-Tau181 antibody contained in the reagent 2 recognize different epitopes of the p-Tau181 protein, respectively, and avoid steric hindrance.

[0021] In order to verify the detection effect of the AD diagnostic kit for detecting the content of p-Tau181 protein by the chemiluminescence method provided by the application, comparative experiments of luminous intensity were carried out with commercially available acridinium ester and luminol products, and comparative experiments of light bleaching and stability were carried out with the markers of acridinium ester and luminol.

[0022] In the comparative experiments of the AD diagnostic kit for detecting the content of p-Tau181 protein by the chemiluminescence method provided by the application and commercially available acridinium ester and luminol products, the full-automatic chemiluminescence analyzer was used as a detection tool, and the luminous intensity was compared.

[0023] The markers in the AD diagnostic kit provided by the application were compared with the markers of acridinium ester and luminol, and light bleaching and stability experiments were carried out.

[0024] Advantages of the application The ABEI macrolide synthesis method provided by the application has a simple process, and by optimizing various experimental parameters, the purity and yield of the ABEI macrolide are significantly improved, providing feasibility for large-scale preparation. The reason why the application uses ABEI macrolide as a marker is based on the structural modification of isoluminol (ABEI), which improves the stability and pi conjugation of the macrolide cyclic structure to enhance the light emission, and improves the chemical stability and intrinsic properties of the marker.

[0025] The application uses ABEI macrolide as a marker, and the chemiluminescence reaction does not need to rely on horseradish peroxidase HRP and traditional enhancers (such as p-iodophenol, luminol enhancer), and the detection system is more simple. One of the advantages is to avoid the risk of HRP dependence: the traditional HRP labeling system is easily disturbed by pH fluctuation, temperature change and inhibitors (such as heavy metal ions and reducing agents), resulting in poor signal stability; the application eliminates such interference through an enzyme-free system, and the signal output is more stable. The second advantage is that the enhancer has a non-specific reaction: the traditional enhancer is easily combined with sample matrix (such as hemoglobin, bilirubin and lipid components in blood) in a non-specific manner, resulting in an increase in background signal; the self-luminous property of ABEI macrolide significantly reduces the background noise and improves the signal-to-noise ratio.

[0026] The AD detection kit of the application detects the p-Tau181 antigen standard, and the p-Tau181 protein presents a good linear relationship (R 2 >0.99) in the concentration range of 0.05-500 pg / mL, which completely covers the clinical detection requirement, and the clinical reference range is usually 1-100 pg / mL.

[0027] The AD detection kit of the application has high sensitivity and low detection limit: the detection limit is as low as 0.9 pg / mL (better than 5-10 pg / mL of the traditional luminol labeling system), which can meet the precise detection requirement of trace p-Tau181 in the cerebrospinal fluid of early Alzheimer's disease patients. Experiments prove that: in terms of light intensity, ABEI macrolide ≈ acridyl ester > luminol; from the light bleaching experiment data, the anti-light bleaching ability of ABEI macrolide is the strongest, which helps the long-term stability of the quality of the reagent; from the stability experiment data, in the accelerated stability experiment and the freeze-thaw stability experiment, the stability of ABEI macrolide is the highest, and the quality stability of the reagent is better. The experimental data show that the light intensity of ABEI macrolide is significantly better than that of the traditional luminol, and is equivalent to that of acridyl ester, but its non-enzyme-dependent and low-background characteristics make up for the pH sensitivity and enhancer interference defects of acridyl ester, and the comprehensive performance reaches the leading level of the chemical luminescence detection field. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the standard curve of the p-Tau181 protein concentration and the light intensity of the application; Figure 2 is the light bleaching experiment data in example 8 of the application; Figure 3 is the data table of the accelerated stability experiment in example 8 of the application; Figure 4 is the data table of the freeze-thaw stability experiment in example 8 of the application; Figure 5 is the molecular structural formula of ABEI macrolide. DETAILED DESCRIPTION

[0029] The application will be described in detail below through examples, the examples of the application are implemented on the premise of the technical scheme of the application, and detailed implementation modes and specific operation processes are given, but the protection scope of the application is not limited to the examples below.

[0030] The p-Tau181 antibody used in the examples of the application is an anti-human p-Tau181 monoclonal antibody.

[0031] Example 1: Preparation of ABEI macrolide

[0032] Since there is no commercially available product of ABEI macrolide at present, the process thereof is optimized based on the synthetic route reported in the literature, and the purity and yield of the target product are significantly improved by regulating the reaction conditions, and the specific steps are as follows: S1. N-(4-aminobutyl)-N-ethyl isorubin (ABEI, 0.1-0.5 mol / L) and glutaric anhydride (0.1-0.6 mol / L) are added to 20 mL of anhydrous pyridine solvent at a molar ratio of 1:1.2-1.5, and stirred at 50°C for 20 min. After the reaction is completed, the mixed solution is concentrated under reduced pressure to remove the solvent, and the remaining crude product is separated and purified by silica gel column chromatography. The eluent used in the separation and purification process is: petroleum ether / ethyl acetate=3:1 v / v, the main fraction is collected and dried under reduced pressure to obtain the ABEI-glutarate intermediate.

[0033] S2. The ABEI-glutarate prepared above is mixed with (benzotriazol-1-yloxy) tripyrrolidinyl phosphonium hexafluorophosphate (PyBOP) at a molar ratio of 1.2-2.0:1 in 25 mL of mixed solvent (DMF / acetonitrile=1:1 v / v), and reacted at 60°C for 120 h; after the reaction is completed, the solvent is removed under reduced pressure, and the remaining crude product is purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate=3:1 v / v), the main fraction is collected and dried, and finally the high-purity ABEI macrolide target product is obtained.

[0034] The preparation method of ABEI macrolide provided by the application can improve the purity of ABEI macrolide to 92%, and the yield to 56%.

[0035] Example 2: Preparation of ABEI macrolide labeled p-Tau181 antibody (1) The p-Tau181 antibody is subjected to dialysis purification treatment: The p-Tau181 antibody is subjected to dialysis treatment with 0.1 M PBS (pH 7.4) buffer (10 kDa dialysis bag is selected, and dialysis is performed at 4°C for 12-24 hours), and the residual ammonium ions and other small molecule interferents in the sample are removed.

[0036] (2) ABEI macrolide and p-Tau181 antibody are subjected to coupling reaction: the molar ratio of ABEI macrolide and p-Tau181 antibody is 20:1; The specific operation of the coupling reaction is as follows: the ABEI macrolide is washed twice by centrifugation in 0.05M MES buffer, pH 5.0; 200uL of MES buffer, 3.5uL of 10mg / mL EDC, and 14uL of 10mg / mL NHS are added, and the carboxyl group is activated in an ice water bath for 20 minutes. After centrifugation, it is re-dispersed in 0.05M MES buffer, pH 5.0; 0.1mL of 10μg / mL p-Tau181 antibody is added, mixed, and reacted at 0°C for 2 hours in the dark. 0.02mL of 1% BSA is added to block the carboxyl activation site for 20-40 minutes, and centrifuged 1-2 times to remove the unreacted p-Tau181 antibody, and re-dispersed in 0.05M MES buffer and stored at 4°C for standby.

[0037] In the above step (2), the process of labeling the p-Tau181 antibody with the ABEI macrolide is to connect the carboxyl group of the ABEI macrolide and the amino group of the p-Tau181 antibody through an amide chemical bond.

[0038] Example 3: Preparation of p-Tau181 antibody labeled magnetic beads 1) The p-Tau181 antibody is dialyzed with 0.1M PBS (pH 7.4), and the operation method is the same as in Example 2.

[0039] 2) Coupling reaction: the mass ratio of p-Tau181 antibody to carboxyl magnetic beads is 1:3.

[0040] 100μL of carboxyl magnetic beads (10mg / mL) are added with 200μL of MES buffer, 7μL of 10mg / mL EDC, and 28μL of 10mg / mL NHS, and the carboxyl group is activated in an ice water bath for 20 minutes; after centrifugation, it is re-dispersed in 0.05M MES buffer, pH 5.0; 0.1mL of 10μg / mL p-Tau181 antibody is added, mixed, and reacted at 0°C for 2 hours in the dark; 0.02mL of 1% BSA is added to block the carboxyl activation site for 20-40 minutes, and centrifuged 1-2 times to remove the unreacted p-Tau181 antibody, and re-dispersed in 0.05M MES buffer and stored at 4°C for standby.

[0041] The p-Tau181 antibody is covalently coupled and fixed on the surface of the magnetic beads, and is used for specific capture of p-Tau181 antigens in the sample.

[0042] The p-Tau181 antibody labeled magnetic beads are a conjugate formed by connecting the amino group of the p-Tau181 antibody and the carboxyl group of the magnetic beads through an amide chemical bond, and are used for specifically capturing the p-Tau181 antigen in a sample; and the ABEI macrolide-labeled p-Tau181 antibody is used for detecting and quantifying the captured p-tau181 protein.

[0043] The coupling amount of the p-Tau181 antibody is 5-20 μg of antibody / mg of magnetic beads, that is, the amount of the coupled p-Tau181 antibody on each milligram of the magnetic beads is between 5 and 20 μg.

[0044] The magnetic beads are superparamagnetic carboxyl magnetic beads with a diameter of 1-5 μm, and the surface is subjected to EDC / NHS activation treatment.

[0045] Example 4: Preparation of p-Tau181 antigen standard and verification of an AD diagnostic kit The purchased p-Tau181 antigen is prepared at a ratio of 0 pg / mL, 0.05 pg / mL, 0.1 pg / mL, 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, and 500 pg / mL for simulating the p-Tau181 antigen in serum, and the specific operation is as follows: The standard buffer solution is 100 mM Tris-HCl with a pH of 8.5, and 11 standard samples with concentration gradients of 0 pg / mL, 0.05 pg / mL, 0.1 pg / mL, 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, and 500 pg / mL are prepared by using the standard buffer solution.

[0046] The present application uses a full-automatic chemiluminescence analyzer as a detection tool, and the methodological mode is a double-antibody sandwich method, that is, different concentrations of p-Tau181 antigen standards are sequentially added to the detection tube of the instrument, 50 uL of a blood sample, 50 uL of reagent 1 (containing p-Tau181 antibody labeled magnetic beads), and 50 uL of reagent 2 (containing ABEI macrolide-labeled p-Tau181 antibody) are added, reaction is performed for 15 min, after magnetic separation and washing, 150 uL of an excitation solution is added, and the test is repeated for three times, and the detection data is shown in Table 1. The luminescence intensity is recorded. The luminescence value is used as the ordinate, and the p-Tau181 concentration is used as the abscissa, and a standard curve is drawn. The standard curve of p-Tau181 is shown in Figure 1 . The linear range (0-500) pg / mL, the correlation coefficient r 2 =0.9972.

[0047] From Figure 1It is evident that the AD diagnostic kit for detecting p-Tau181 protein content using chemiluminescence immunoassay provided by this invention exhibits good linearity within the range of p-Tau181 antigen standard concentration from 0.05 to 500 pg / mL, meeting the clinical p-Tau181 antigen detection range. It also demonstrates high signal-to-noise ratio and sensitivity, with a detection limit of 0.9 pg / mL, exhibiting excellent sensing performance.

[0048] Table 1: Luminescence intensity of antigen standards / RLU Concentration pg / mL RLU1 RLU2 RLU3 Average luminescence intensity / RLU 0 37 49 43 43 0.05 89 96 103 96 0.1 150 159 144 151 0.5 732 399 456 529 1 1001 1520 1854 1458 5 10965 14872 15623 13820 10 16230 15396 16451 16026 50 103655 85753 105224 98211 100 125487 135489 154664 138547 500 645890 639255 640834 641993 Example 5: Detection Limit Experiment The specific measurement steps are as follows: Measure the luminescence intensity (RLU) of at least 20 blank samples (0 pg / mL) to obtain the blank signal dataset, as shown in Table 2 below.

[0049] Table 2: Blank Signal Dataset 37 49 43 34 40 46 49 46 42 39 50 36 34 38 45 48 41 36 50 51 2) Calculate the mean (RLU0) and standard deviation (SD0) of the blank signal; Based on the blank signal dataset, the following values ​​were calculated: RLU0 mean = 42.7, standard deviation SD0 = 5.61; 3) Usually, k=3 is chosen (corresponding to a 99.7% confidence interval, i.e., the 3-standard-deviation method) to obtain the critical RLU value (RLU). 临界 =RLU0+3×SD0). Based on the above experimental data, the RLU can be derived. 临界 =RLU0 mean + 3 × SD0 = 59.53; 4) Combining the standard curve (the fitting equation for concentration-RLU), deduce the relationship with RLU. 临界 The corresponding concentration is the limit of detection (LOD); the LOD is as low as 0.9 pg / mL.

[0050] Example 6: Preparation of Activation Solution The excitation solution contains Tris-HCl and H2O2. Because the rate and intensity of the chemiluminescence reaction are highly sensitive to pH and require precise control, the following pH ranges were set for Tris-HCl: 7.5, 8.0, 8.5, 9.0, and 9.5. Since the buffer concentration affects the ionic strength and buffering capacity of the system, the concentration range of Tris-HCl is 50 mM, 100 mM, and 150 mM. The specific screening process is as follows: the preferred Tris-HCl has a pH of 8.5 and a concentration of 100 mM.

[0051] 1) Add 50 pg / mL of p-Tau181 standard, 50 μL of blood sample, 50 μL of reagent 1 (magnetic beads labeled with p-Tau181 antibody), and 50 μL of reagent 2 (p-Tau181 antibody labeled with ABEI macrolide) to the instrument detection tube in sequence, and react for 15 min; after magnetic separation and washing, add 150 μL of excitation solution (100 mM Tris-HCl pH=7.5) and record the luminescence intensity.

[0052] 2) Unlike 1), 150 μL of excitation solution (100 mM Tris-HCl pH=8.0) was used to record the luminescence intensity.

[0053] 3) Unlike 1), 150 μL of excitation solution (100 mM Tris-HCl pH=8.5) was used to record the luminescence intensity.

[0054] 4) Unlike 1), 150 μL of excitation solution (100 mM Tris-HCl pH=9.0) was used to record the luminescence intensity.

[0055] 5) Unlike 1), 150 μL of excitation solution (100 mM Tris-HCl pH=9.5) was used to record the luminescence intensity.

[0056] 6) Unlike 1), 150 μL of excitation solution (50 mM Tris-HCl pH=8.5) was used to record the luminescence intensity.

[0057] 7) Unlike 1), use 150 μL of excitation solution (100 mM Tris-HCl pH=8.5) and record the luminescence intensity.

[0058] 8) Unlike 1), use 150 μL of excitation solution (150 mM Tris-HCl pH=8.5) and record the luminescence intensity.

[0059] The effect of changes in conditions on the excitation solution in the AD diagnostic kit provided by this invention on the detection results (luminescence intensity) was verified by experiments. The experimental results are shown in Table 3 below: Table 3: Effect of excitation solution on luminescence intensity Serial number Conditions Luminescence intensity / RLU 1 100 mM Tris-HCl pH=7.5 35120 2 100 mM Tris-HCl pH=8.0 56284 3 100 mM Tris-HCl pH=8.5 100826 4 100 mM Tris-HCl pH=9.0 62485 5 100 mM Tris-HCl pH=9.5 36121 6 50 mM Tris-HCl pH=8.5 83125 7 100 mM Tris-HCl pH=8.5 106284 8 150 mM Tris-HCl pH=8.5 100826 Example 7

[0060] To verify the detection efficacy of the AD diagnostic kit for detecting p-Tau181 protein content using chemiluminescence immunoassay provided by this invention, a comparative experiment on luminescence intensity was conducted with commercially available acridinium ester and luminol products. In the comparative experiments, a fully automated chemiluminescence analyzer was used as the detection tool to compare the luminescence intensity.

[0061] Acridinium ester products (using industry-standard products, specific manufacturers not listed) include the following reagents: Reagent A: Magnetic bead-labeled capture antibody, Reagent B: Acridinium ester-labeled detection antibody, Reagent C: Reaction buffer solution Reagent D: Chemiluminescent excitation solution.

[0062] Luminescence assay of acridinium ester products: p-Tau181 standard (three different concentrations of 0.1 pg / mL, 50 pg / mL, and 500 pg / mL were added in portions) sequentially to the instrument detection tube, along with 50 μL of blood sample, reagent A, reagent B, and reagent C. The mixture was reacted for 15 min, followed by magnetic separation and washing. Then, reagent D was added, and the luminescence intensity was recorded. The assay was repeated three times with three different concentrations of p-Tau181 standard (0.1 pg / mL, 50 pg / mL, and 500 pg / mL).

[0063] Luminol products (using industry-standard products, specific manufacturers not listed) include the following reagents: Reagent A: Magnetic bead-labeled capture antibody, Reagent B: Horseradish peroxidase (HRP) labeled detection antibody Reagent C: Reaction buffer Reagent D: Luminescent excitation solution Luminescence assay for luminol products: p-Tau181 standard (three different concentrations of 0.1 pg / mL, 50 pg / mL, and 500 pg / mL were added in portions) sequentially to the instrument detection tube, along with 50 μL of blood sample, reagent A, reagent B, and reagent C. The mixture was reacted for 15 min, followed by magnetic separation and cleaning. Then, reagent D was added, and the luminescence intensity was recorded. The test was repeated three times.

[0064] The luminescence assay of the diagnostic kit (ABEI macrolide product) of this invention: p-Tau181 standard (added in portions at three different concentrations: 0.1 pg / mL, 50 pg / mL, and 500 pg / mL), 50 μL of blood sample, 50 μL of reagent 1 (containing p-Tau181 antibody-labeled magnetic beads), and 50 μL of reagent 2 (containing ABEI macrolide-labeled p-Tau181 antibody) were added sequentially to the instrument detection tube, and the reaction was allowed to proceed for 15 min. After magnetic separation and washing, 150 μL of excitation buffer (100 mM Tris-HCl, pH = 8.5) was added, and the luminescence intensity was recorded. The test was repeated 3 times.

[0065] The comparison data of luminescence intensity between the diagnostic kit of this invention (ABEI macrolide product) and commercially available acridine ester and luminol products are shown in Table 4 below: Table 4: Comparison of Luminous Intensity RLU Concentration pg / mL Acridinium ester product RLU Luminol product RLU ABEI macrolide product RLU 0.1 123 56 150 0.1 129 53 159 0.1 102 46 144 50 99025 10252 103655 50 82565 13467 95753 50 81264 20584 105224 500 535153 51851 645890 500 482562 41554 639255 500 465821 46922 640834 As can be seen from the data in the chart, the luminescence intensity of the diagnostic kit of this invention (ABEI macrolide product) is greater than that of the acridinium ester product and the luminol product.

[0066] Example 8: Photobleaching comparison experiment and stability experiment To illustrate the photobleaching and stability of the detection reagents in the AD diagnostic kit provided by this invention, photobleaching and stability experiments were conducted on ABEI macrolide label (i.e., ABEI macrolide), acridine ester label (N-methylacridin-N'-succinimide ester, CAS number 115853-74-2), and luminol label (3-aminophthalic acid hydrazide, CAS number 521-31-3).

[0067] (a) Photobleaching comparison experiment Commercially available acridil ester and luminol labels were used. Following the standard CLSI EP25 Guideline for Stability Assessment of In Vitro Diagnostic Reagents, photobleaching validation experimental conditions were set up, using a xenon lamp with an irradiance of 0.51 W / (m²·nm)@340 nm and an illumination time of 24 h. The initial and final values ​​of the luminescence signal intensity of the labels were recorded, and the retention rate of luminescence intensity was calculated.

[0068] From Table 5 (Appendix) Figure 2 Based on photobleaching data, ABEI macrolide has the strongest resistance to photobleaching, which contributes to the long-term stability of reagent quality.

[0069] (II) Stability Comparison Experiment: 1) Commercially available acridinium ester and luminol labeling were used, referring to the "Guiding Principles for Registration Review of Stability Studies of In Vitro Diagnostic Reagents (2025)".

[0070] 2) Set up an accelerated stability test (store at 37℃, and take samples at 1 day, 3 days, 1 week, 2 weeks, and 1 month. Record the initial and final values ​​of the luminescence intensity of the marker, and calculate the rate of decrease in luminescence intensity. The deviation of the marker's luminescence signal intensity from the initial value is ≤15%).

[0071] 3) Set up a freeze-thaw stability test (freeze at -20℃ → thaw at room temperature, repeat 3-5 cycles, no precipitation / layering. Record the initial and final values ​​of the luminescence intensity of the marker, and calculate the rate of decrease in luminescence intensity). Prepare the acridinium ester, luminol, and ABEI macrolide concentrations according to the table below.

[0072] 4) In the accelerated stability test, see Figure 3Based on experimental data, acridine esters showed a 50% decrease in signal within 3 days, luminol a 30% decrease within 1 month, and ABEI macrolides a <10% decrease within 1 month, indicating that ABEI macrolides have the highest stability.

[0073] See Figure 4 In the freeze-thaw stability test, the signal of acridine ester decreased by 40% after 3 cycles, the signal of luminol decreased by 25% after 3 cycles, and the signal of ABEI macrolide decreased by <15% after 5 cycles. ABEI macrolide has the highest stability.

Claims

1. A method of labeling ABEI macrocyclic lactone p-Tau181 antibodies, characterized by, Comprising the following steps: (1) The p-Tau181 antibody is subjected to dialysis purification treatment to fully remove the residual ammonium ions and other small molecule interferents in the sample; (2) ABEI macrolide and p-Tau181 antibody are subjected to coupling reaction: the molar ratio of ABEI macrolide and p-Tau181 antibody is 20:1; The specific operation of the coupling reaction is as follows: ABEI macrolide is washed twice by centrifugation using 0.05M MES buffer, pH 5.0; 200uL of MES buffer, 3.5uL of 10mg / mL EDC, and 14uL of 10mg / mL NHS are added, and the carboxyl group is activated in an ice water bath for 20 minutes; after centrifugation, it is re-dispersed in 0.05M MES buffer, pH 5.0; 0.1mL of 10ug / mL p-Tau181 antibody is added, mixed, and reacted at 0℃ for 2 hours in the dark; 0.02mL of 1% BSA is added to block the carboxyl activation site for 20-40 minutes, and the unreacted p-Tau181 antibody is removed by centrifugation and re-dispersed in 0.05M MES buffer for storage at 4℃.

2. The method of ABEI macrolide-labeled p-Tau181 antibody according to claim 1, characterized in that, In the above step (2), the process of labeling p-Tau181 antibody with ABEI macrolide is to connect the carboxyl group of ABEI macrolide and the amino group of p-Tau181 antibody through an amide chemical bond.

3. The method of ABEI macrolide-labeled p-Tau181 antibody according to claim 1 or 2, characterized in that, The ABEI macrolide is prepared by the following steps: S1. Commercially available N-(4-aminobutyl)-N-ethyl isorumi is added to anhydrous pyridine solvent at a molar ratio of 1:1.2-1.5, and the mixture is magnetically stirred at 50℃ for 20 minutes. After the reaction is completed, column chromatography is used for purification to obtain the ABEI-glutarate intermediate; S2. The prepared ABEI-glutarate intermediate is added to a mixed solvent of DMF / acetonitrile=1:1 v / v at a molar ratio of 1.2-2.0:1, and the mixture is reacted at 60℃ for 120 hours. After the reaction is completed, column chromatography is used for purification to obtain the ABEI macrolide.

4. An AD diagnostic kit for detecting the content of p-Tau181 protein by a chemiluminescence method, characterized by, The following reagents are included: Reagent 1, containing p-Tau18 antibody labeled magnetic beads; Reagent 2, containing ABEI macrolide labeled p-Tau181 antibody; p-Tau181 antigen standard and stimulating solution; The reagent 1 includes p-Tau18 antibody labeled magnetic beads, contains bovine serum albumin BSA, sodium azide, and PBS buffer with a pH value of 7.4; The reagent 2 includes ABEI macrolide labeled p-Tau181 antibody, contains Tris-HCl PH=8.0, ProClin300 buffer; The p-Tau181 antigen standard includes 11 standard samples with p-Tau181 protein concentrations of 0 pg / mL, 0.05 pg / mL, 0.1 pg / mL, 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, and 500 pg / mL. The excitation solution contains Tris-HCl and H2O2, preferably Tris-HCl with a pH of 8.5 and a concentration of 100 mM, and H2O2 with a content of 0.1-0.5% (v / v).

5. The AD diagnostic kit according to claim 4, characterized by The p-Tau181 antibody-labeled magnetic beads are a conjugate formed by the amino group of the p-Tau181 antibody and the carboxyl group of the magnetic beads through an amide chemical bond, and are used for specific capture of p-Tau181 antigens in samples; and the ABEI macrolide-labeled p-Tau181 antibody is used for detection and quantification of the captured p-tau181 protein.

6. The AD diagnostic kit according to claim 5, characterized by The coupling amount of the p-Tau181 antibody is 5-20 μg of antibody per mg of magnetic beads; and the magnetic beads are superparamagnetic carboxyl magnetic beads with a diameter of 1-5 μm, and the surface is activated by EDC / NHS.

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