Method and kit for improving phosphorylated tau protein detection sensitivity

Abstract

The invention relates to the field of phosphorylated tau protein detection, in particular to a method for improving phosphorylated tau protein detection sensitivity and a kit thereof. The invention provides a method for improving phosphorylated tau protein detection sensitivity. The method comprises the following steps: S101, obtaining a blood sample; s102, adding the detection reagent into the blood sample; s103, detecting the concentration of the phosphorylated tau protein in the blood sample by using a chemiluminescence method; wherein the detection reagent comprises an alkaline phosphatase polymer-antibody compound. The alkaline phosphatase polymer-antibody compound prepared by the invention is high in yield, good in stability, high in coupling efficiency, low in background, good in precision, strong in anti-interference capability, smaller in batch difference and lower in non-specific adsorption, the sensitivity is remarkably improved, and the compound shows remarkable advantages in detection of phosphorylated tau protein in a blood sample.

Description

Technical Field

[0001] The present invention relates to the field of phosphorylated tau protein detection, and in particular to a method for improving the sensitivity of phosphorylated tau protein detection and a kit thereof. Background Art

[0002] Alzheimer's disease (AD) is a highly insidious and progressive neurological disease with a high prevalence among the elderly, and the number of patients with mild cognitive impairment (MCI) has reached 38.77 million. AD is the most common dementia, and its typical characteristics are early impairments in learning, short-term memory, and sensory orientation. It is also associated with the gradual loss of complex attention, language, executive function, self-care ability, and changes in emotions, thinking, and behavior, which seriously endangers human health.

[0003] Neuropathological hallmarks of AD include extracellular amyloid beta (Aβ) plaque deposition and intracellular neurofibrillary tangles (NFTs), which are mainly composed of aggregated tau protein and hyperphosphorylated tau protein. Currently, cerebrospinal fluid (CSF) and PET / CT detection are the gold standard for AD diagnosis, but due to the high cost of detection, high sampling risk, poor patient compliance and poor accessibility, large-scale promotion is difficult. Compared with normal people, the concentrations of ptau181 and ptau217 proteins in the blood samples of AD patients are significantly increased, and they are well consistent with the significant Aβ concentration and tau-PET positivity. Therefore, the changes in the concentrations of ptau181 and ptau217 proteins in the blood are used for individualized prediction of AD patients and mild cognitive impairment (MCI) patients, which has very important clinical value. However, due to the existence of a blood-brain barrier in the human brain, the concentrations of markers such as phosphorylated tau protein in the blood are very low, so the detection of phosphorylated tau protein is still mainly based on CSF samples.

[0004] Blood testing mainly uses mass spectrometry, single molecule detection (Simoa technology) and chemiluminescence as the main detection methods. If the test object with extremely low content is to be detected, higher requirements are placed on the sensitivity of the detection method. Mass spectrometry has high requirements for equipment and is expensive. Simoa technology has poor precision and expensive consumables, so its promotion is relatively limited. Chemiluminescence detection is relatively easy to promote and has a wider audience due to its characteristics of simple automatic operation, good repeatability and low detection cost. However, the sensitivity of conventional chemiluminescence detection is not as good as single molecule detection, and the detection of low-abundance markers is still a great challenge. Summary of the invention

[0005] In a first aspect, the present invention provides a method for improving the sensitivity of phosphorylated tau protein detection, characterized in that the method comprises the following steps: S101 obtaining a blood sample, wherein the blood sample contains phosphorylated tau protein; S102 adding a detection reagent to the blood sample; S103 detecting the concentration of the phosphorylated tau protein in the blood sample using a chemiluminescence method; The detection reagent comprises an alkaline phosphatase polymer-antibody complex, wherein the alkaline phosphatase polymer comprises a polymer backbone and an alkaline phosphatase connected to the polymer backbone via a coupling agent, an antibody is coupled to the alkaline phosphatase polymer, the polymer backbone is PAMAM, the antibody specifically recognizes the phosphorylated tau protein, and the coupling agent comprises SM (PEG) n ; wherein the molecular weight of the PAMAM is selected from 3-30 kDa, and n is selected from an integer of 0-8.

[0006] In some embodiments, the phosphorylated tau protein includes one or more of ptau181, ptau217, ptau231, and ptau205.

[0007] In some embodiments, the phosphorylated tau protein is ptau181 and / or ptau217.

[0008] In some embodiments, the blood sample comprises the phosphorylated tau protein at a concentration of at least 0.1 pg / mL.

[0009] In some embodiments, the blood sample comprises the phosphorylated tau protein at a concentration of ≥ 1 pg / mL.

[0010] In some embodiments, the antibody is a Fab fragment or a F(ab')2 fragment, preferably a F(ab')2 fragment.

[0011] In some embodiments, the detection reagent further comprises a magnetic particle reagent coated with a phosphorylated tau protein antibody.

[0012] As used herein, "SMCC (Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 4-(N-maleimidomethyl)cyclohexane-1-carboxylate)" refers to a succinimide ester having amine reactivity. N A cross-linking reagent with bifunctional groups of -hydroxysuccinimide (NHS ester) group and thiol-reactive maleimide group. It can react with amino group and thiol group to form stable covalent bonds, and in this way, it can connect molecules containing amino group and molecules containing thiol group together.

[0013] As used herein, "SMCC-PEG (or SM(PEG) n )" refers to SMCC cross-linking reagents with a polyethylene glycol spacer arm (usually containing n equal to 0 to 24 ethylene glycol units).

[0014] As used herein, "coupling" includes direct or indirect connection of two or more moieties.

[0015] In some embodiments, the SM(PEG) n Includes one or more of SM(PEG)2, SM(PEG)4, SM(PEG)6 and SM(PEG)8.

[0016] As used herein, "PAMAM" refers to polyamidoamine dendrimers. They are synthesized by a divergent method, where polymerization occurs gradually from the initiating core (ethylenediamine) outward, and each cycle of reaction adds one layer or one generation to the already formed polymer, thereby forming dendrimers of different generations, denoted as G1, G2, etc. PAMAM molecules begin to have a spherical structure from G4, with a hydrophobic cavity inside.

[0017] In some embodiments, the PAMAM is a third generation PAMAM, a fourth generation PAMAM, or a fifth generation PAMAM.

[0018] In some embodiments, the PAMAM has a molecular weight of 6-30 kDa.

[0019] In some embodiments, the concentration of the alkaline phosphatase polymer-antibody complex comprises 0.1 - 5 μg / mL.

[0020] In some embodiments, the step S103 further comprises adding a chemiluminescent substrate.

[0021] In some embodiments, the method for preparing the alkaline phosphatase polymer-antibody complex comprises the following steps: S201: mixing and reacting the activated polymer backbone and the alkaline phosphatase activated by the first coupling agent, and then adding a blocking agent to terminate the reaction to obtain an alkaline phosphatase polymer; S202 activating the alkaline phosphatase polymer with a second coupling agent to obtain an activated alkaline phosphatase polymer; S203 mixing and reacting the activated antibody and the activated alkaline phosphatase polymer to obtain the alkaline phosphatase polymer-antibody complex; Wherein, the blocking agent includes cysteine ​​and N-(2-aminoethyl)maleimide.

[0022] In some embodiments, the molar ratio of cysteine ​​to N-(2-aminoethyl)maleimide is 1:10-4:5. In some embodiments, the final molar number of cysteine ​​added comprises 200 nmoL, and the final molar number of N-(2-aminoethyl)maleimide added comprises 500 nmoL.

[0023] In some embodiments, the polymer backbone is selected from PAMAM.

[0024] In some embodiments, the PAMAM is a third generation PAMAM, a fourth generation PAMAM, or a fifth generation PAMAM.

[0025] In some embodiments, the activated polymer backbone is activated by an activating agent.

[0026] In some embodiments, the molar ratio of the activator to the number of amino groups in the polymer backbone (feed ratio) is 0.5:1-2:1.

[0027] In some embodiments, the activating agent comprises 2-iminothiolane hydrochloride.

[0028] In some embodiments, the first coupling agent comprises SM(PEG) n .

[0029] In some embodiments, the second coupling agent comprises SM(PEG) n .

[0030] In some embodiments, n is selected from an integer of 0-8.

[0031] In some embodiments, the SM(PEG) n Includes one or more of SM(PEG)2, SM(PEG)4, SM(PEG)6 and SM(PEG)8.

[0032] In some embodiments, the molar ratio (feed ratio) of the alkaline phosphatase polymer to the second coupling agent is 1:10-15.

[0033] In some embodiments, the activated antibody is activated by the activating agent.

[0034] In some embodiments, the antibody is a Fab fragment or a F(ab')2 fragment, preferably a F(ab')2 fragment.

[0035] In some embodiments, the antibody specifically recognizes the phosphorylated tau protein.

[0036] In some embodiments, the molar ratio (feed ratio) of the activator to the antibody is 5:1-20:1.

[0037] In some embodiments, the mass ratio of the activated antibody to the activated alkaline phosphatase polymer comprises 1:1.

[0038] In a second aspect, the present invention provides a detection reagent, characterized in that the detection reagent comprises an alkaline phosphatase polymer-antibody complex, wherein the alkaline phosphatase polymer comprises a polymer backbone and alkaline phosphatase connected to the polymer backbone through a coupling agent, the alkaline phosphatase polymer is coupled to an antibody, the polymer backbone is PAMAM, and the coupling agent comprises SM (PEG) n , the antibody specifically recognizes the phosphorylated tau protein; wherein the molecular weight of the PAMAM is selected from 3-30 kDa, and n is selected from an integer of 0-8.

[0039] In some embodiments, the phosphorylated tau protein includes one or more of ptau181, ptau217, ptau231, and ptau205.

[0040] In some embodiments, the phosphorylated tau protein is ptau181 and / or ptau217.

[0041] In some embodiments, the detection reagent is used to detect a blood sample.

[0042] In some embodiments, the blood sample comprises the phosphorylated tau protein at a concentration of at least 0.1 pg / mL.

[0043] In some embodiments, the blood sample comprises the phosphorylated tau protein at a concentration of ≥ 1 pg / mL.

[0044] In some embodiments, the antibody is a Fab fragment or a F(ab')2 fragment, preferably a F(ab')2 fragment.

[0045] In some embodiments, the method for preparing the alkaline phosphatase polymer-antibody complex comprises the following steps: S201: mixing and reacting a polymer backbone activated by an activating agent and an alkaline phosphatase activated by a first coupling agent, and then adding a blocking agent to terminate the reaction to obtain an alkaline phosphatase polymer; S202 activating the alkaline phosphatase polymer with a second coupling agent to obtain an activated alkaline phosphatase polymer; S203 mixing and reacting the activated antibody and the activated alkaline phosphatase polymer to obtain the alkaline phosphatase polymer-antibody complex; Wherein, the blocking agent includes cysteine ​​and N-(2-aminoethyl)maleimide.

[0046] In some embodiments, the molar ratio of the cysteine ​​to the N-(2-aminoethyl)maleimide is 1:10-4:5.

[0047] In some embodiments, the final molar amount of the cysteine ​​added comprises 200 nmoL, and the final molar amount of the N-(2-aminoethyl)maleimide added comprises 500 nmoL.

[0048] In some embodiments, the polymer backbone is selected from PAMAM.

[0049] In some embodiments, the PAMAM is a third generation PAMAM, a fourth generation PAMAM, or a fifth generation PAMAM.

[0050] In some embodiments, the molar ratio of the activator to the number of amino groups in the polymer backbone (feed ratio) is 0.5:1-2:1.

[0051] In some embodiments, the activating agent comprises 2-iminothiolane hydrochloride.

[0052] In some embodiments, the first coupling agent comprises SM(PEG) n .

[0053] In some embodiments, the second coupling agent comprises SM(PEG) n .

[0054] In some embodiments, n is selected from an integer of 0-8.

[0055] In some embodiments, the SM(PEG) n Includes one or more of SM(PEG)2, SM(PEG)4, SM(PEG)6 and SM(PEG)8.

[0056] In some embodiments, the molar ratio (feed ratio) of the alkaline phosphatase polymer to the second coupling agent is 1:10-15.

[0057] In some embodiments, the activated antibody is activated by the activating agent.

[0058] In some embodiments, the antibody is a Fab fragment or a F(ab')2 fragment, preferably a F(ab')2 fragment.

[0059] In some embodiments, the antibody specifically recognizes the phosphorylated tau protein.

[0060] In some embodiments, the molar ratio (feed ratio) of the activator to the antibody is 5:1-20:1.

[0061] In some embodiments, the mass ratio of the activated antibody to the activated alkaline phosphatase polymer comprises 1:1.

[0062] In a third aspect, the present invention also provides a kit for detecting phosphorylated tau protein, which comprises the above-mentioned detection reagent.

[0063] In a fourth aspect, the present invention provides SM(PEG) n Application in the preparation of a reagent for testing phosphorylated tau protein, wherein n is an integer of 2-8, and the SM (PEG) n Used as a coupling agent.

[0064] In some embodiments, the reagent for detecting phosphorylated tau protein comprises an alkaline phosphatase polymer-antibody complex.

[0065] In some embodiments, the alkaline phosphatase polymer comprises a polymer backbone and alkaline phosphatase linked to the polymer backbone via the coupling agent, and the alkaline phosphatase polymer is coupled to an antibody.

[0066] In some embodiments, the antibody specifically recognizes the phosphorylated tau protein.

[0067] In some embodiments, the polymer backbone is PAMAM.

[0068] In some embodiments, the PAMAM has a molecular weight of 3-30 kDa.

[0069] In a fifth aspect, the present invention provides the use of cysteine ​​and N-(2-aminoethyl)maleimide in the preparation of a reagent for testing phosphorylated tau protein, wherein the molar ratio of the cysteine ​​to the N-(2-aminoethyl)maleimide is 1:10-4:5.

[0070] In some embodiments, the reagent for detecting phosphorylated tau protein comprises an alkaline phosphatase polymer-antibody complex.

[0071] In some embodiments, the alkaline phosphatase polymer comprises a polymer backbone and alkaline phosphatase connected to the polymer backbone via a coupling agent, and an antibody is coupled to the alkaline phosphatase polymer, wherein the alkaline phosphatase polymer is obtained by mixing and reacting an activated polymer backbone and alkaline phosphatase activated by a first coupling agent, and then adding a blocking agent to terminate the reaction, and the cysteine ​​and the N-(2-aminoethyl)maleimide are used as blocking agents.

[0072] In some embodiments, the polymer backbone is PAMAM.

[0073] In some embodiments, the antibody specifically recognizes the phosphorylated tau protein.

[0074] In some embodiments, the PAMAM has a molecular weight of 3-30 kDa.

[0075] In some embodiments, the first coupling agent comprises SM(PEG) n .

[0076] In some embodiments, n is selected from an integer of 0-8.

[0077] In a sixth aspect, the present invention provides SM(PEG) n and / or cysteine ​​and N-(2-aminoethyl)maleimide in the preparation of a reagent for testing phosphorylated tau protein, characterized in that n is an integer of 2-8, the SM (PEG) n Used as a coupling agent; the molar ratio of the cysteine ​​to the N-(2-aminoethyl)maleimide is 1:10-4:5.

[0078] In some embodiments, the reagent for detecting phosphorylated tau protein comprises an alkaline phosphatase polymer-antibody complex.

[0079] In some embodiments, the alkaline phosphatase polymer comprises a polymer backbone and alkaline phosphatase linked to the polymer backbone via the coupling agent, and the alkaline phosphatase polymer is coupled to an antibody.

[0080] In some embodiments, the polymer backbone is PAMAM.

[0081] In some embodiments, the antibody specifically recognizes the phosphorylated tau protein.

[0082] In some embodiments, the PAMAM has a molecular weight of 3-30 kDa.

[0083] In some embodiments, the alkaline phosphatase polymer is obtained by mixing and reacting an activated polymer backbone and alkaline phosphatase activated with a first coupling agent, and then adding a blocking agent to terminate the reaction, and the cysteine ​​and the N-(2-aminoethyl)maleimide are used as blocking agents.

[0084] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects: In order to achieve protein polymerization and thus improve sensitivity, the prior art generally uses dextran, chitosan, bovine serum albumin, PAMAM and other materials as polymer skeletons for polymerization treatment. For example, Chinese patent application CN112305222A discloses a small polymer enzyme-antibody fragment, which uses a smaller dendritic polymer skeleton (such as G1PAMAM) to make the enzyme connected to the skeleton more compact and occupy less space, thereby having better tissue penetration ability for application in the field of immunohistochemistry. There are currently no reports on the use of alkaline phosphatase polymer-antibody complexes to detect phosphorylated tau protein in blood samples.

[0085] The inventors of the present invention have found that although conventional alkaline phosphatase polymer-antibody complexes have a large number of active groups and more active sites within the molecule, their complex structure and the presence of active groups seriously reduce their storage stability. When they are applied to chemiluminescence detection, they are prone to serious nonspecific adsorption, inevitably causing false positive problems, making it difficult to achieve a real sensitivity improvement.

[0086] The present invention selects PAMAM with a suitable molecular weight, and simultaneously uses SM (PEG) containing PEG with a suitable chain length in the process of using it to polymerize alkaline phosphatase. n As a coupling agent. Unexpectedly, the present invention found that the yield, water solubility and stability of the alkaline phosphatase polymer prepared by the present invention are improved, while the enzyme activity of alkaline phosphatase can be well maintained (even improved), and the non-specific adsorption problem of conventional alkaline phosphatase polymers is overcome (and the background is reduced), which is further reflected in the significantly enhanced sensitivity, precision and anti-interference ability when detecting phosphorylated tau protein in blood samples.

[0087] In addition, the present invention controls the type of blocking agent (specifically, cysteine ​​carries a free amino group, which can be used for the next round of labeling reaction, and cysteine ​​as an amphoteric amino acid can also increase the water solubility of the polymer; N-(2-aminoethyl)maleimide blocks the remaining thiol groups, and the amino group it carries can also be used for subsequent labeling reactions), the ratio of the alkaline phosphatase polymer and the coupling agent, and synergistically ensures the stable molecular weight and degree of polymerization (i.e., good uniformity) of the prepared alkaline phosphatase polymer. On the basis of significantly improved sensitivity, the batch difference control is also more accurate, avoiding the problem of inaccurate results caused by batch difference fluctuations at high sensitivity.

[0088] In addition, when coupled with the F(ab')2 fragment, the F(ab')2 fragment not only does not affect the sensitivity, stability, uniformity and other properties of the alkaline phosphatase polymer prepared by the present invention, but synergistically reduces nonspecific interference, improves the precision of the reagent, and improves the sensitivity to the picogram level. The embodiments of the present invention show that when the alkaline phosphatase polymer-antibody complex prepared by the present invention is used to detect phosphorylated tau protein in blood samples, the sensitivity can reach 0.4 pg / mL (even 0.1 pg / mL), which is not only suitable for detecting phosphorylated tau protein in blood samples of AD patients, but also suitable for detecting extremely low levels of phosphorylated tau protein in blood samples of MCI patients, so it can better monitor the development of AD.

[0089] In summary, the alkaline phosphatase polymer-antibody complex prepared by the present invention has high yield, good stability, high coupling efficiency, significantly improved sensitivity, low background, good precision, strong anti-interference ability, smaller batch difference and lower nonspecific adsorption, showing significant advantages in detecting phosphorylated tau protein in blood samples. DETAILED DESCRIPTION

[0090] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0091] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0092] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0093] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0094] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0095] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0096] Example 1: Preparation of alkaline phosphatase polymer 1.1 Screening of PAMAMs with different molecular weights In this example, PAMAM (NH2) is selected 16 (i.e. G2) (molecular weight 3.2 KDa), PAMAM (NH2) 32 (i.e. G3) (molecular weight 6.9 KDa), PAMAM (NH2) 64 (i.e. G4) (molecular weight 14.2 KDa), PAMAM (NH2) 128 (i.e. G5) (molecular weight 28.8KDa), PAMAM (NH2) 256 (i.e. G6) (molecular weight 58.0 KDa) five different molecular weight PAMAMs were used to prepare alkaline phosphatase polymers, and their yields and stabilities were compared. The specific preparation methods are as follows: ① Branched chain activation: Take the corresponding volume of branched chain concentrate according to the number of amino groups in 50 nmoL of PAMAM (G2~G6) with different molecular weight branches, and then add activation buffer with pH 7.2~8.0 to dilute to 0.2 mL for use. Select 2-iminothiolane hydrochloride (2IT) as the branched chain activator and prepare the concentrate at a concentration of 5 mg / mL. According to the molar ratio of 2IT: amino number of 0.5:1~2:1, take the corresponding volume of 2IT concentrate (5 mg / mL) and add it to the diluted branched chain solution above, mix well, react at 25℃ for 1h, and then desalt; ②ALP activation: 1.0 mg of ALP was taken separately, desalted using a desalting column, and then the concentration was adjusted to 4 mg / mL using an activation buffer. The desalted ALP was mixed with SMCC at a molar ratio of 1:10, mixed and reacted at 25°C for 1 hour, and then desalted; ③ALP polymerization: Mix the activated desalted side chain with the activated desalted ALP in a molar ratio of 1:2, mix well and react at 25°C for 1 hour, then add 200 nmoL of 2-MEA (2-mercaptoethylamine hydrochloride) to terminate for 30 minutes, and then add 500 nmoL of iodoacetamide to terminate for 30 minutes; ④ Purification: The ALP polymer mixture is filtered and purified by molecular exclusion chromatography, and the purified products are collected according to different collection peaks, stored at 2-8°C, and the corresponding yield is calculated simultaneously; the yield is calculated as follows: yield = (amount of purified product of polymerized ALP / theoretical amount of polymerized ALP) × 100%; ⑤ Comparison of the stability of polymerized ALP prepared by PAMAM with different molecular weights: The purified products of polymerized ALP prepared by PAMAM with different molecular weights (alkaline phosphatase polymers) were prepared at 1 μg / mL, respectively, and placed in a 37°C oven for acceleration for 0, 3, 7, and 12 days. After the acceleration, 10 μL was taken and added to the substrate to verify the stability of the alkaline phosphatase polymer before and after acceleration. The stability is reflected by calculating the increase in the light value on the nth day of acceleration, where the calculation method for the increase in the light value on the nth day of acceleration is: the increase in the light value on the nth day of acceleration = (light value on the nth day of acceleration - light value on the 0th day / light value on the 0th day) × 100%.

[0097] The results are shown in Table 1. This example found that if the molecular weight of the PAMAM side chain is too high (e.g., G6 generation and above) or too low (e.g., G2 generation and below), the yield of the alkaline phosphatase polymer will be reduced. Therefore, this example preliminarily selected G3, G4, and G5 PAMAM as the polymer backbone to prepare the alkaline phosphatase polymer.

[0098] Table 1: Comparison of the yields of alkaline phosphatase polymers prepared from PAMAM with different molecular weights The results are shown in Table 2. Whether it is accelerated for 3 days, 7 days or 12 days, the accelerated decline of the alkaline phosphatase polymer prepared by G2-G5 PAMAM can be controlled within 5%, meeting the stability requirements. However, the accelerated decline of the alkaline phosphatase polymer prepared by G6 PAMAM after 12 days is close to 10%, which may be due to the fact that the alkaline phosphatase polymer prepared by G6 PAMAM is too large, resulting in a decrease in stability.

[0099] Table 2: Comparison of the stability of alkaline phosphatase polymers prepared from PAMAM with different molecular weights 1.2 Optimization of nonspecific adsorption of alkaline phosphatase polymers In this embodiment, five coupling agents with different chain lengths (i.e., SMCC, SM(PEG)2, SM(PEG)4, SM(PEG)8, SM(PEG) 12 ) to prepare alkaline phosphatase polymers and compare their yields and nonspecific adsorption. The specific preparation method is as follows: ① Branched chain activation: Select G5 PAMAM according to the number of amino groups of 50 nmoL, take the corresponding volume of branched chain concentrate, and then add the activation buffer of pH 7.2~8.0 to dilute to 0.2 mL for use. Select 2-iminothiolane hydrochloride (2IT) as the branched chain activator and prepare the concentrate at a concentration of 5 mg / mL. According to the molar ratio of 2IT: amino group number of 1:1, measure the corresponding volume of 2IT concentrate (5 mg / mL) and add it to the diluted branched chain solution above, mix well, react at 25℃ for 1h, and then desalt; ②ALP activation: 1.0 mg of ALP was taken and desalted using a desalting column, and then the concentration was adjusted to 4 mg / mL using activation buffer. The desalted ALP was activated using SMCC, SM(PEG)2, SM(PEG)4, SM(PEG)8, SM(PEG) 12 The mixture was mixed at a molar ratio of 1:10, reacted at 25°C for 1 hour, and then desalted; ③ALP polymerization: Mix the activated desalted side chain with the activated desalted ALP in a molar ratio of 1:2, and react at 25°C for 1 hour. Add 200 nmoL of 2-MEA to terminate the reaction for 30 minutes, and then add 500 nmoL of iodoacetamide to terminate the reaction for 30 minutes; ④ Purification: The ALP polymer mixture was filtered and purified by molecular exclusion chromatography, the purified product was collected and stored at 2-8°C, and the corresponding yield was calculated simultaneously; ⑤ Comparison of non-specific adsorption of polymerized ALP activated by coupling agents of different chain lengths: The purified products of polymerized ALP activated by coupling agents of different chain lengths were prepared into 1 μg / mL working solutions, and detected with antibody-coated magnetic beads. After incubating 30 μL of 1 mg / mL antibody-coated magnetic beads with 60 μL of polymerized ALP working solution for 10 minutes, the detection was repeated 10 times on the chemiluminescence instrument, and the background difference was calculated and compared; at the same time, 10 μL of polymerized ALP working solution was added to the substrate to verify whether there was a difference in the light value; The results are shown in Table 3. This example found that appropriately extending the chain length of the coupling agent can further improve the yield of the alkaline phosphatase polymer, while the excessive length of the coupling agent leads to a decrease in the yield. Therefore, this example preferably uses SM(PEG)2-8 as a coupling agent (activator) to prepare the alkaline phosphatase polymer.

[0100] Table 3: Effects of coupling agents of different chain lengths on the yield of alkaline phosphatase polymers The results are shown in Table 4. This example found that the coupling agent containing a PEG spacer arm (i.e., SM(PEG) n ) can not only increase the yield of alkaline phosphatase polymers, but also significantly reduce the nonspecific adsorption of alkaline phosphatase polymers to magnetic beads while maintaining or even increasing the enzyme activity of alkaline phosphatase. 12 Although it can reduce the nonspecific adsorption to magnetic beads, it may reduce the enzymatic activity of alkaline phosphatase due to the excessive chain length. Based on this, SM (PEG) was selected in this embodiment after comprehensive consideration. 2-8 (e.g. SM(PEG)2, SM(PEG)4, SM(PEG)8) as coupling agents (activators) to prepare alkaline phosphatase polymers.

[0101] Table 4: Nonspecific adsorption of ALP polymerized by coupling agents (activators) of different chain lengths to magnetic beads Embodiment 2 2.1 Preparation of F(ab')2 fragment antibodies: ① Take 1 mg of tau protein mouse monoclonal antibody, replace the antibody with pH 2.5 citrate buffer through a desalting column, and adjust the concentration to 4 mg / mL for later use; ② Dissolve pepsin powder in pH 2.5 citric acid buffer to 4 mg / mL for later use; ③ Mix the antibody and pepsin at a mass ratio of 5:1, mix well and place in a 37°C oven for reaction for 2 hours; ④ Adjust the pH of the reaction solution to 8.0 to terminate the enzyme digestion reaction; ⑤ Purification: Filter and purify through molecular sieves, collect the obtained F(ab')2 fragments, adjust to the corresponding concentration and store at 2-8°C for future use; 2.2 Blocking optimization of alkaline phosphatase polymer ① Branched chain activation: Select G5 PAMAM according to the number of amino groups of 50 nmoL, take the corresponding volume of branched chain concentrate, and then add the activation buffer of pH 7.2~8.0 to dilute to 0.2 mL for use. Select 2-iminothiolane hydrochloride (2IT) as the branched chain activator and prepare the concentrate at a concentration of 5 mg / mL. According to the molar ratio of 2IT: amino group number of 1:1, measure the corresponding volume of 2IT concentrate (5 mg / mL) and add it to the diluted branched chain solution above, mix well, react at 25℃ for 1h, and then desalt; ②ALP activation: 1.0 mg of ALP was taken separately, desalted using a desalting column, and then the concentration was adjusted to 4 mg / mL using an activation buffer. The desalted ALP was mixed with SM(PEG)4 at a molar ratio of 1:10, mixed and reacted at 25°C for 1 hour, and then desalted; ③ALP polymerization: The activated desalted side chain and the activated desalted ALP were mixed in a molar ratio of 1:2, and the mixture was reacted at 25°C for 1 hour. One group was terminated by adding 200 nmoL of 2-MEA at a final molar number for 30 minutes, and then adding 500 nmoL of iodoacetamide at a final molar number for 30 minutes; the other group was terminated by adding 200 nmoL of cysteine ​​at a final molar number for 30 minutes, and then adding 500 nmoL of N-(2-aminoethyl)maleimide at a final molar number for 30 minutes; ④Purification: The ALP polymer mixture was filtered and purified by size exclusion chromatography, and the purified product was collected and adjusted to a concentration of 4 mg / mL and stored at 2-8°C; ⑤ F(ab')2 activation: Take 0.5 mg of the F(ab')2 fragment antibody after enzyme digestion and adjust the concentration to 2 mg / mL. Select 2-iminothiolane hydrochloride (2IT) as the activator and prepare a concentrated solution at a concentration of 5 mg / mL. According to the molar ratio of 2IT: F(ab')2 of 5:1, measure the corresponding volume of 2IT concentrated solution and add it to the above F(ab')2, mix well, react at 25°C for 1 hour, and then desalt; ⑥ Activation of polymerized ALP: 0.5 mg of the polymerized ALP collected by the above different termination methods was taken and activated according to the molar ratio of polymerized ALP: SM (PEG) 4 of 1:10. After mixing, the mixture was reacted at 25°C for 1 hour, and then desalted; ⑦ Coupling of F(ab')2 and polymerized ALP: according to the mass ratio of 1:1, the above activated F(ab')2 and activated polymerized ALP were measured, mixed and reacted at 25°C for 1h; ⑧Purification: The F(ab')2 and polymerized ALP conjugate was filtered and purified by molecular exclusion chromatography, the purified product was collected and stored at 2-8°C, the corresponding yield was calculated simultaneously, and the effects of two different blocking agent combinations on the coupling efficiency of F(ab')2 and polymerized ALP were compared.

[0102] The results are shown in Table 5. This example found that using cysteine ​​and N-(2-aminoethyl)maleimide as blocking agents can improve the coupling efficiency of F(ab')2 and polymerized ALP. The blocking agent combination of cysteine ​​and N-(2-aminoethyl)maleimide is beneficial to the subsequent re-activation coupling (i.e., the coupling of F(ab')2 and polymerized ALP), and the yield of alkaline phosphatase polymer-antibody complex is higher.

[0103] Table 5: Effects of two different combinations of blocking agents on coupling efficiency 2.3 Preparation of F(ab')2 fragment antibody and polymerized ALP conjugate (alkaline phosphatase polymer-antibody complex) ① Branched chain activation: Select G5 PAMAM according to the number of amino groups of 50 nmoL, take the corresponding volume of branched chain concentrate, and then add the activation buffer of pH 7.2~8.0 to dilute to 0.2 mL for use. Select 2-iminothiolane hydrochloride (2IT) as the branched chain activator and prepare the concentrate at a concentration of 5 mg / mL. According to the molar ratio of 2IT: amino group number of 1:1, take the corresponding volume of 2IT concentrate (5 mg / mL) and add it to the diluted branched chain solution above, mix well, react at 25℃ for 1h, and then desalt; ②ALP activation: 1.0 mg of ALP was taken separately, desalted using a desalting column, and then the concentration was adjusted to 4 mg / mL using an activation buffer. The desalted ALP was mixed with SM(PEG)4 at a molar ratio of 1:10, mixed and reacted at 25°C for 1 hour, and then desalted; ③ALP polymerization: Mix the activated desalted branched chain with the activated desalted ALP in a molar ratio of 1:2, and react at 25°C for 1 hour. Add 200 nmoL of cysteine ​​to terminate the reaction for 30 minutes, and then add 500 nmoL of N-(2-aminoethyl)maleimide to terminate the reaction for 30 minutes; ④Purification: The ALP polymer mixture was filtered and purified by size exclusion chromatography, and the purified product was collected and adjusted to a concentration of 4 mg / mL and stored at 2-8°C; ⑤ F(ab')2 activation: Take 0.5 mg of F(ab')2 fragment antibody of tau protein after enzyme digestion and adjust the concentration to 2 mg / mL. Select 2-iminothiolane hydrochloride (2IT) as the activator and prepare a concentrated solution at a concentration of 5 mg / mL. According to the molar ratio of 2IT: F(ab')2 of 5:1, measure the corresponding volume of 2IT concentrated solution and add it to the above F(ab')2, mix well, react at 25°C for 1 hour, and then desalt; ⑥ Activation of polymerized ALP: 0.5 mg of the collected polymerized ALP was added to the mixture at a molar ratio of 1:10 for polymerized ALP:SM(PEG)4, mixed well, reacted at 25°C for 1 hour, and then desalted; ⑦ Coupling of F(ab')2 and polymerized ALP: according to the mass ratio of 1:1, the above activated F(ab')2 and activated polymerized ALP were measured, mixed and reacted at 25°C for 1h; ⑧Purification: The F(ab')2 and polymerized ALP conjugates are filtered and purified by size exclusion chromatography, and the purified products are collected and stored at 2-8°C; 2.4 Performance verification of the conjugate of F(ab')2 fragment antibody and polymerized ALP The F(ab')2 fragment antibodies used in this example are all prepared by enzymatic digestion of tau protein mouse monoclonal antibodies. This example mainly verifies the relevant performance by detecting two items (typical phosphorylated tau proteins) ptau181 and ptau217. The reagents tested in this example are as follows: Reagent 1: Component ①: ptau181-specific antibody-coated magnetic beads 1 mg / mL, Component ②: Tau-F(ab')2-polymerized ALP working solution 0.5 μg / mL; Reagent 2: Component ①: ptau217-specific antibody-coated magnetic beads 1 mg / mL, Component ②: Tau-F(ab')2-polymerized ALP working solution 0.5 μg / mL; Reagent 3: Component ①: ptau181-specific antibody-coated magnetic beads 1 mg / mL, Component ②: Tau-F(ab')2-ALP working solution 0.5 μg / mL; Reagent 4: Component ①: ptau217-specific antibody-coated magnetic beads 1 mg / mL, Component ②: Tau-F(ab')2-ALP working solution 0.5 μg / mL; Reagent 5: Component ①: ptau181-specific antibody-coated magnetic beads 1 mg / mL, Component ②: Tau-IgG-polymerized ALP working solution 0.5 μg / mL; Reagent 6: Component ①: ptau217-specific antibody-coated magnetic beads 1 mg / mL, Component ②: Tau-IgG-polymerized ALP working solution 0.5 μg / mL; Reaction mode: Add 50 μL of antigen working solution of different concentrations, add 30 μL of component ①, incubate for 10 minutes and then wash. After washing, add 60 μL of component ② and incubate for 10 minutes. After the reaction is completed, wash and add substrate for light measurement.

[0104] 2.4.1 Sensitivity Verification Use reagents 1-4 to test the antigen working solution of corresponding concentration series respectively, repeat the test twice at each concentration point, calculate the average value of each point, and compare the corresponding signal-to-noise ratio S / N and background; 2.4.2 Precision Verification By adding high-concentration antigen ptau181 to normal human plasma samples, low- and high-concentration samples with concentrations of 1 pg / mL and 4 pg / mL were prepared. Similarly, by adding high-concentration antigen ptau217 to normal human plasma samples, low- and high-concentration samples with concentrations of 0.4 pg / mL and 4 pg / mL were prepared, and the samples were tested 10 times, and the mean, standard deviation and corresponding coefficient of variation (CV) were calculated. The coefficient of variation was calculated as follows: CV = standard deviation / mean × 100%; 2.4.3 Anti-interference capability verification Five human anti-mouse antibody (HAMA) and five rheumatoid factor (RF) positive plasmas were selected and tested according to the reaction mode to compare the improvement of anti-interference ability; 2.4.4 Thermal Acceleration Stability Verification The reagents were placed in a 37°C oven for accelerated storage for 0, 7, and 12 days, and the stability before and after the acceleration was compared.

[0105] The results are shown in Table 6. This example found that the results of detecting ptau181 or ptau217 in the blood showed that the sensitivity of the conjugate of the F(ab')2 fragment antibody and / or polymerized ALP prepared by the present invention was significantly improved, the signal-to-noise ratio (S / N) could be increased by about 4 to 5 times, and the background was relatively lower.

[0106] Table 6: Sensitivity comparison The results are shown in Table 7. Compared with Reagent 3 and Reagent 4, the intra-batch coefficient of variation of the conjugate of the F(ab')2 fragment antibody and multimerized ALP prepared by the present invention (i.e., Reagent 1 and Reagent 2) is smaller, which may be due to the better uniformity of the conjugate of the F(ab')2 fragment antibody and multimerized ALP prepared by the present invention, thereby effectively improving the precision.

[0107] Table 7: Precision comparison The results are shown in Table 8. This example found that compared with the conjugate prepared using IgG (reagent 5 and reagent 6), the conjugate of the F(ab')2 fragment antibody prepared by the present invention and polymerized ALP (i.e., reagent 1 and reagent 2) had lower detection results for ptau181 and ptau217, and showed significantly enhanced anti-interference ability when detecting HAMA and RF positive samples, which can reduce the cost of using blocking agents.

[0108] Table 8: The results are shown in Tables 9 and 10. Compared with Reagent 3 and Reagent 4, the conjugates of the F(ab')2 fragment antibody prepared by the present invention and polymerized ALP (i.e., Reagent 1 and Reagent 2) showed lower thermal acceleration 7-day and 12-day declines in the detection of ptau181 and ptau217, and the overall declines were within 6.2%, meeting the stability requirements.

[0109] Table 9: Thermal Acceleration Stability Verification Table 10: Thermal Acceleration Stability Verification In summary, the alkaline phosphatase polymer-antibody complex prepared by the present invention has high yield, good stability, high coupling efficiency, significantly improved sensitivity (the sensitivity in the two projects of ptau181 and ptau217 is improved by 4 to 5 times), lower background, good precision, and strong anti-interference ability (for example, anti-HAMA and RF interference), which can reduce the cost of using blockers, and at the same time, has smaller batch difference and lower nonspecific adsorption, showing significant advantages in detecting phosphorylated tau protein in blood samples.

[0110] The embodiments of the present invention are described above, but the present invention is not limited to the above-mentioned specific implementation modes. The above-mentioned specific implementation modes are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which are all within the protection of the present invention.

Claims

1. A method for improving the sensitivity of phosphorylated tau protein detection, characterized in that: The method comprises the following steps: S101 obtaining a blood sample, wherein the blood sample contains phosphorylated tau protein; S102 adding a detection reagent to the blood sample; S103 detecting the concentration of the phosphorylated tau protein in the blood sample using a chemiluminescence method; The detection reagent comprises an alkaline phosphatase polymer-antibody complex, wherein the alkaline phosphatase polymer comprises a polymer backbone and an alkaline phosphatase connected to the polymer backbone via a coupling agent, an antibody is coupled to the alkaline phosphatase polymer, the polymer backbone is PAMAM, the antibody specifically recognizes the phosphorylated tau protein, and the coupling agent comprises SM (PEG) n ; wherein the molecular weight of the PAMAM is selected from 3-30 kDa, and n is selected from an integer of 0-8.

2. The method according to claim 1, characterized in that The antibody is a Fab fragment or a F(ab')2 fragment, preferably a F(ab')2 fragment.

3. The method according to claim 1, characterized in that The preparation method of the alkaline phosphatase polymer-antibody complex comprises the following steps: S201: mixing and reacting the activated polymer backbone and the alkaline phosphatase activated by the first coupling agent, and then adding a blocking agent to terminate the reaction to obtain an alkaline phosphatase polymer; S202 activating the alkaline phosphatase polymer with a second coupling agent to obtain an activated alkaline phosphatase polymer; S203 mixing and reacting the activated antibody and the activated alkaline phosphatase polymer to obtain the alkaline phosphatase polymer-antibody complex; Wherein, the blocking agent includes cysteine ​​and N-(2-aminoethyl)maleimide.

4. The method according to claim 3, characterized in that The first coupling agent and the second coupling agent include SM (PEG) n .

5. The method according to claim 4, characterized in that The molar ratio of the alkaline phosphatase polymer to the second coupling agent is 1:10-15.

6. A detection reagent, characterized in that: The detection reagent comprises an alkaline phosphatase polymer-antibody complex, wherein the alkaline phosphatase polymer comprises a polymer backbone and alkaline phosphatase connected to the polymer backbone via a coupling agent, an antibody is coupled to the alkaline phosphatase polymer, the polymer backbone is PAMAM, and the coupling agent comprises SM (PEG) n , the antibody specifically recognizes the phosphorylated tau protein; wherein the molecular weight of the PAMAM is selected from 3-30 kDa, and n is selected from an integer of 0-8.

7. The detection reagent according to claim 6, characterized in that The antibody is a Fab fragment or a F(ab')2 fragment, preferably a F(ab')2 fragment.

8. The detection reagent according to claim 6 or 7, characterized in that The preparation method of the alkaline phosphatase polymer-antibody complex comprises the following steps: S201: mixing and reacting the activated polymer backbone and the alkaline phosphatase activated by the first coupling agent, and then adding a blocking agent to terminate the reaction to obtain an alkaline phosphatase polymer; S202 activating the alkaline phosphatase polymer with a second coupling agent to obtain an activated alkaline phosphatase polymer; S203 mixing and reacting the activated antibody and the activated alkaline phosphatase polymer to obtain the alkaline phosphatase polymer-antibody complex; Wherein, the blocking agent includes cysteine ​​and N-(2-aminoethyl)maleimide; preferably, the second coupling agent includes SM (PEG) n .

9. The detection reagent according to claim 8, characterized in that The molar ratio of the alkaline phosphatase polymer to the second coupling agent is 1:10-15.

10. A kit for detecting phosphorylated tau protein, comprising the detection reagent according to any one of claims 6 to 9.

11. SM(PEG) n and / or use of cysteine ​​and N-(2-aminoethyl)maleimide in the preparation of a reagent for testing phosphorylated tau protein, characterized in that: The molar ratio of the cysteine ​​to the N-(2-aminoethyl)maleimide is 1:10-4:5, and n is an integer of 2-8.

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