Method for improving the sensitivity of detection of phosphorylated tau protein and kit thereof

Abstract

The present application relates to the field of phosphorylated tau protein detection, and particularly relates to a method for improving the sensitivity of phosphorylated tau protein detection and a kit thereof.The present application provides a method for improving the sensitivity of phosphorylated tau protein detection, comprising: S101 obtaining a blood sample; S102 adding a detection reagent to the blood sample; S103 detecting the concentration of phosphorylated tau protein in the blood sample using a chemiluminescence method; wherein the detection reagent comprises an alkaline phosphatase polymer-antibody complex.The alkaline phosphatase polymer-antibody complex prepared by the present application has high yield, good stability, high coupling efficiency, significantly improved sensitivity, low background, good precision, strong anti-interference ability, smaller batch difference and lower non-specific adsorption, and exhibits significant advantages in detecting phosphorylated tau protein in blood samples.

Description

Technical Field

[0001] This invention relates to the field of phosphorylated tau protein detection, specifically to a method and kit for improving the sensitivity of phosphorylated tau protein detection. Background Technology

[0002] Alzheimer's disease (AD) is a highly insidious and progressive neurological disorder with a high prevalence among the elderly, with as many as 38.77 million people experiencing mild cognitive impairment (MCI). Alzheimer's disease is the most common form of dementia, characterized by early impairment in learning, short-term memory, and sensory orientation. It is also associated with progressive loss of complex attention, language, executive function, self-care abilities, and changes in mood, thinking, and behavior, seriously jeopardizing human health.

[0003] Neuropathological markers of Alzheimer's disease (AD) include extracellular β-amyloid (Aβ) plaque deposition and intracellular neurofibrillary tangles (NFTs), primarily composed of aggregated and hyperphosphorylated tau protein. Currently, cerebrospinal fluid (CSF) and PET / CT scans are the gold standard for AD diagnosis; however, their high cost, high sampling risk, poor patient compliance, and limited accessibility make large-scale implementation difficult. Compared to healthy individuals, AD patients show significantly elevated concentrations of ptau181 and ptau217 proteins in their blood samples, exhibiting strong concordance with Aβ concentrations and tau-PET positivity. Therefore, changes in blood ptau181 and ptau217 protein concentrations have significant clinical value for individualized prediction in AD and mild cognitive impairment (MCI) patients. However, due to the blood-brain barrier, the concentration of phosphorylated tau proteins and other markers in the blood is very low; therefore, CSF samples remain the primary method for detecting phosphorylated tau proteins.

[0004] Blood tests primarily utilize mass spectrometry, single-molecule detection (SimoA technology), and chemiluminescence immunoassay as the main methods. Detecting analytes at extremely low concentrations places higher demands on the sensitivity of the detection method. Mass spectrometry requires sophisticated and expensive equipment, while SimoA technology suffers from poor precision and high consumable costs, thus limiting its widespread adoption. Chemiluminescence immunoassay, on the other hand, is easier to implement and has a wider audience due to its fully automated operation, good repeatability, and low cost. However, conventional chemiluminescence immunoassays are less sensitive than single-molecule detection, and detecting low-abundance labeled analytes remains a significant challenge. Summary of the Invention

[0005] In a first aspect, the present invention provides a method for improving the detection sensitivity of phosphorylated tau protein, characterized in that the method includes the following steps:

[0006] S101 Obtain a blood sample, wherein the blood sample contains phosphorylated tau protein;

[0007] S102 Add the test reagent to the blood sample;

[0008] S103 The concentration of phosphorylated tau protein in the blood sample was detected using a chemiluminescence method;

[0009] The detection reagent comprises an alkaline phosphatase polymer-antibody complex. The alkaline phosphatase polymer includes a polymer backbone and an alkaline phosphatase linked 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. The coupling agent includes SM(PEG). n The molecular weight of PAMAM is selected from 3-30 kDa, and n is selected from integers from 0-8.

[0010] In some embodiments, the phosphorylated tau protein includes one or more of ptau181, ptau217, ptau231, and ptau205.

[0011] In some embodiments, the phosphorylated tau protein is ptau181 and / or ptau217.

[0012] In some embodiments, the blood sample contains a phosphorylated tau protein at a concentration of at least 0.1 pg / mL.

[0013] In some embodiments, the blood sample contains a concentration of phosphorylated tau protein ≥1 pg / mL.

[0014] In some embodiments, the antibody is a Fab fragment or an F(ab')2 fragment, preferably an F(ab')2 fragment.

[0015] In some embodiments, the detection reagent further includes magnetic microparticle reagents coated with phosphorylated tau protein antibodies.

[0016] As used in this article, "SMCC (Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 4-(N-maleimidomethyl)cyclohexane-1-carboxylate)" refers to amine-reactive succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. NA crosslinking agent with bifunctional groups of hydroxysuccinimide (NHS ester) and thiol-reactive maleimide. It can react with amino and thiol groups respectively to form stable covalent bonds, thereby linking molecules containing amino groups and molecules containing thiol groups together.

[0017] As used in this article, "SMCC-PEG (or SM(PEG)" n ")" refers to an SMCC crosslinking agent with polyethylene glycol spacer arms (typically containing n equal to 0 to 24 ethylene glycol units).

[0018] The term "coupling" as used in this article includes direct or indirect connections between two or more parts.

[0019] In some embodiments, the SM(PEG) n Including one or more of SM(PEG)2, SM(PEG)4, SM(PEG)6 and SM(PEG)8.

[0020] As used in this article, "PAMAM" refers to polyamide-amine dendritic polymers. They are synthesized via a divergent polymerization method, where polymerization occurs progressively from the initiating core (ethylenediamine). Each reaction cycle adds one layer or generation to the already formed polymer, resulting in dendritic polymers of different generations, denoted as G1, G2, etc. From G4 onwards, PAMAM molecules exhibit a spherical structure with internal hydrophobic cavities.

[0021] In some embodiments, the PAMAM is a third-generation PAMAM, a fourth-generation PAMAM, or a fifth-generation PAMAM.

[0022] In some embodiments, the molecular weight of the PAMAM is 6-30 kDa.

[0023] In some embodiments, the concentration of the alkaline phosphatase polymer-antibody complex includes 0.1–5 μg / mL.

[0024] In some embodiments, step S103 further includes adding a chemiluminescent substrate.

[0025] In some embodiments, the method for preparing the alkaline phosphatase polymer-antibody complex includes the following steps:

[0026] S201 involves mixing and reacting the activated polymer backbone with alkaline phosphatase activated by the first coupling agent, then adding a blocking agent to terminate the reaction, thereby obtaining an alkaline phosphatase polymer.

[0027] S202 The alkaline phosphatase polymer is activated with a second coupling agent to obtain an activated alkaline phosphatase polymer;

[0028] S203 The activated antibody and the activated alkaline phosphatase polymer are mixed and reacted to obtain the alkaline phosphatase polymer-antibody complex;

[0029] The blocking agent includes cysteine ​​and N-(2-aminoethyl)maleimide.

[0030] In some embodiments, the molar ratio of cysteine ​​to N-(2-aminoethyl)maleimide is 1:10-4:5. In some embodiments, the final molar amount of cysteine ​​added includes 200 nmol, and the final molar amount of N-(2-aminoethyl)maleimide added includes 500 nmol.

[0031] In some embodiments, the polymer backbone is selected from PAMAM.

[0032] In some embodiments, the PAMAM is a third-generation PAMAM, a fourth-generation PAMAM, or a fifth-generation PAMAM.

[0033] In some embodiments, the activated polymer backbone is activated by an activator.

[0034] In some embodiments, the molar ratio (feed ratio) of the activator to the number of amino groups in the polymer backbone includes 0.5:1 to 2:1.

[0035] In some embodiments, the activator comprises 2-iminothione hydrochloride.

[0036] In some embodiments, the first coupling agent comprises SM(PEG). n .

[0037] In some embodiments, the second coupling agent comprises SM(PEG). n .

[0038] In some embodiments, n is selected from integers from 0 to 8.

[0039] In some embodiments, the SM(PEG) n Including one or more of SM(PEG)2, SM(PEG)4, SM(PEG)6 and SM(PEG)8.

[0040] In some embodiments, the molar ratio (feed ratio) of the alkaline phosphatase polymer to the second coupling agent includes 1:10-15.

[0041] In some embodiments, the activated antibody is activated by the activator.

[0042] In some embodiments, the antibody is a Fab fragment or an F(ab')2 fragment, preferably an F(ab')2 fragment.

[0043] In some embodiments, the antibody specifically recognizes the phosphorylated tau protein.

[0044] In some embodiments, the molar ratio (feed ratio) of the activator to the antibody includes 5:1 to 20:1.

[0045] In some embodiments, the mass ratio of the activated antibody to the activated alkaline phosphatase polymer is 1:1.

[0046] Secondly, 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 an alkaline phosphatase linked to the polymer backbone by 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 PAMAM is selected from 3-30 kDa, and n is selected from integers from 0-8.

[0047] In some embodiments, the phosphorylated tau protein includes one or more of ptau181, ptau217, ptau231, and ptau205.

[0048] In some embodiments, the phosphorylated tau protein is ptau181 and / or ptau217.

[0049] In some embodiments, the test reagent is used to test blood samples.

[0050] In some embodiments, the blood sample contains a phosphorylated tau protein at a concentration of at least 0.1 pg / mL.

[0051] In some embodiments, the blood sample contains a concentration of phosphorylated tau protein ≥1 pg / mL.

[0052] In some embodiments, the antibody is a Fab fragment or an F(ab')2 fragment, preferably an F(ab')2 fragment.

[0053] In some embodiments, the method for preparing the alkaline phosphatase polymer-antibody complex includes the following steps:

[0054] S201 involves mixing and reacting a polymer backbone activated with an activating agent and an alkaline phosphatase activated with a first coupling agent, then adding a blocking agent to terminate the reaction, thereby obtaining an alkaline phosphatase polymer.

[0055] S202 The alkaline phosphatase polymer is activated with a second coupling agent to obtain an activated alkaline phosphatase polymer;

[0056] S203 The activated antibody and the activated alkaline phosphatase polymer are mixed and reacted to obtain the alkaline phosphatase polymer-antibody complex;

[0057] The blocking agent includes cysteine ​​and N-(2-aminoethyl)maleimide.

[0058] In some embodiments, the molar ratio of cysteine ​​to N-(2-aminoethyl)maleimide is 1:10-4:5.

[0059] In some embodiments, the final molar amount of the added cysteine ​​is 200 nmol, and the final molar amount of the added N-(2-aminoethyl)maleimide is 500 nmol.

[0060] In some embodiments, the polymer backbone is selected from PAMAM.

[0061] In some embodiments, the PAMAM is a third-generation PAMAM, a fourth-generation PAMAM, or a fifth-generation PAMAM.

[0062] In some embodiments, the molar ratio (feed ratio) of the activator to the number of amino groups in the polymer backbone includes 0.5:1 to 2:1.

[0063] In some embodiments, the activator comprises 2-iminothione hydrochloride.

[0064] In some embodiments, the first coupling agent comprises SM(PEG). n .

[0065] In some embodiments, the second coupling agent comprises SM(PEG). n .

[0066] In some embodiments, n is selected from integers from 0 to 8.

[0067] In some embodiments, the SM(PEG) n Including one or more of SM(PEG)2, SM(PEG)4, SM(PEG)6 and SM(PEG)8.

[0068] In some embodiments, the molar ratio (feed ratio) of the alkaline phosphatase polymer to the second coupling agent includes 1:10-15.

[0069] In some embodiments, the activated antibody is activated by the activator.

[0070] In some embodiments, the antibody is a Fab fragment or an F(ab')2 fragment, preferably an F(ab')2 fragment.

[0071] In some embodiments, the antibody specifically recognizes the phosphorylated tau protein.

[0072] In some embodiments, the molar ratio (feed ratio) of the activator to the antibody includes 5:1 to 20:1.

[0073] In some embodiments, the mass ratio of the activated antibody to the activated alkaline phosphatase polymer is 1:1.

[0074] Thirdly, the present invention also provides a kit for detecting phosphorylated tau protein, which comprises the above-described detection reagents.

[0075] Fourthly, the present invention provides SM(PEG). n In the preparation of reagents for testing phosphorylated tau protein, n is an integer from 2 to 8, and the SM(PEG) is used. n Used as a coupling agent.

[0076] In some embodiments, the reagent for detecting phosphorylated tau protein includes an alkaline phosphatase polymer-antibody complex.

[0077] In some embodiments, the alkaline phosphatase polymer comprises a polymer backbone and an alkaline phosphatase linked to the polymer backbone via the coupling agent, wherein an antibody is coupled to the alkaline phosphatase polymer.

[0078] In some embodiments, the antibody specifically recognizes the phosphorylated tau protein.

[0079] In some embodiments, the polymer backbone is PAMAM.

[0080] In some embodiments, the molecular weight of the PAMAM is 3-30 kDa.

[0081] Fifthly, the present invention provides the use of cysteine ​​and N-(2-aminoethyl)maleimide in the preparation of reagents for testing phosphorylated tau protein, wherein the molar ratio of cysteine ​​to N-(2-aminoethyl)maleimide is 1:10-4:5.

[0082] In some embodiments, the reagent for detecting phosphorylated tau protein includes an alkaline phosphatase polymer-antibody complex.

[0083] In some embodiments, the alkaline phosphatase polymer comprises a polymer backbone and an alkaline phosphatase linked to the polymer backbone by a coupling agent, wherein 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 an alkaline phosphatase activated with a first coupling agent, followed by the addition of a blocking agent to terminate the reaction, wherein cysteine ​​and N-(2-aminoethyl)maleimide are used as blocking agents.

[0084] In some embodiments, the polymer backbone is PAMAM.

[0085] In some embodiments, the antibody specifically recognizes the phosphorylated tau protein.

[0086] In some embodiments, the molecular weight of the PAMAM is 3-30 kDa.

[0087] In some embodiments, the first coupling agent comprises SM(PEG). n .

[0088] In some embodiments, n is selected from integers from 0 to 8.

[0089] In a sixth aspect, the present invention provides SM(PEG). n The use of cysteine ​​and / or N-(2-aminoethyl)maleimide in the preparation of reagents for testing phosphorylated tau protein, characterized in that n is an integer from 2 to 8, and the SM(PEG) n Used as a coupling agent; the molar ratio of the cysteine ​​and the N-(2-aminoethyl)maleimide is 1:10-4:5.

[0090] In some embodiments, the reagent for detecting phosphorylated tau protein includes an alkaline phosphatase polymer-antibody complex.

[0091] In some embodiments, the alkaline phosphatase polymer comprises a polymer backbone and an alkaline phosphatase linked to the polymer backbone via the coupling agent, wherein an antibody is coupled to the alkaline phosphatase polymer.

[0092] In some embodiments, the polymer backbone is PAMAM.

[0093] In some embodiments, the antibody specifically recognizes the phosphorylated tau protein.

[0094] In some embodiments, the molecular weight of the PAMAM is 3-30 kDa.

[0095] In some embodiments, the alkaline phosphatase polymer is obtained by mixing and reacting an activated polymer backbone with an alkaline phosphatase activated with a first coupling agent, followed by the addition of a blocking agent to terminate the reaction, wherein the cysteine ​​and the N-(2-aminoethyl)maleimide are used as blocking agents.

[0096] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects:

[0097] To achieve protein polymerization and thus improve sensitivity, existing technologies typically use materials such as dextran, chitosan, bovine serum albumin, and PAMAM as polymer backbones for polymerization. For example, Chinese patent application CN112305222A discloses a small polymeric enzyme-antibody fragment that uses a smaller dendritic polymer backbone (e.g., G1PAMAM), making the enzyme attached to the backbone more compact, occupying less space, and thus having better tissue penetration capabilities for application in immunohistochemistry. Currently, there are no reports of using alkaline phosphatase polymeric antibody complexes to detect phosphorylated tau protein in blood samples.

[0098] The inventors of this invention have discovered that although conventional alkaline phosphatase polymer-antibody complexes contain a large number of active groups and have more active sites, their complex structure and the presence of active groups seriously reduce their storage stability. When applied to chemiluminescence detection methods, they are prone to causing serious non-specific adsorption, inevitably leading to false positives and making it difficult to achieve a true improvement in sensitivity.

[0099] This invention selects PAMAM with an appropriate molecular weight and simultaneously uses SM (PEG) containing PEG of appropriate chain length during the alkaline phosphatase polymerization process. n As a coupling agent, this invention unexpectedly found that the yield, water solubility, and stability of the alkaline phosphatase polymer prepared by this invention were all improved, while the enzyme activity of alkaline phosphatase was well maintained (or even improved). It also overcame the non-specific adsorption problem of conventional alkaline phosphatase polymers (and reduced the background). This is further reflected in the significantly enhanced sensitivity, precision, and anti-interference ability when detecting phosphorylated tau protein in blood samples.

[0100] Furthermore, by controlling the type of blocking agent (specifically, cysteine ​​carries a free amino group, which can be used in the next round of labeling reaction, and cysteine, as an amphoteric amino acid, can simultaneously increase the water solubility of the polymer; N-(2-aminoethyl)maleimide blocks the remaining thiol groups, and its own amino group can also be used in subsequent labeling reactions) and the ratio of alkaline phosphatase polymer to coupling agent, this invention 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, batch-to-batch variation control is also more precise, avoiding the problem of inaccurate results caused by batch-to-batch variation fluctuations under high sensitivity.

[0101] Furthermore, when coupled with the F(ab')2 fragment, the F(ab')2 fragment not only does not affect the sensitivity, stability, or uniformity of the alkaline phosphatase polymer prepared in this invention, but also synergistically reduces non-specific interference, improving reagent precision while increasing sensitivity to the picogram level. Examples of this invention show that when using the alkaline phosphatase polymer-antibody complex prepared in this invention to detect phosphorylated tau protein in blood samples, the sensitivity can reach 0.4 pg / mL (even as low as 0.1 pg / mL). This is suitable not only for detecting phosphorylated tau protein in blood samples from AD patients, but also for detecting extremely low levels of phosphorylated tau protein in blood samples from MCI patients, thus enabling better monitoring of AD progression.

[0102] In summary, the alkaline phosphatase polymer-antibody complex prepared by this invention exhibits high yield, good stability, high coupling efficiency, significantly improved sensitivity, low background, good precision, strong anti-interference ability, smaller batch-to-batch variation, and lower non-specific adsorption, demonstrating significant advantages in detecting phosphorylated tau protein in blood samples. Detailed Implementation

[0103] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0104] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0105] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0106] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0107] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0108] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges 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., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0109] Example 1: Preparation of alkaline phosphatase polymer

[0110] 1.1 Screening of PAMAMs with different molecular weights

[0111] In this embodiment, 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 28.8 kDa), PAMAM(NH2) 256 Alkaline phosphatase polymers were prepared from five different molecular weight PAMAMs (G6, molecular weight 58.0 kDa), and their yields and stability were compared. The specific preparation methods are as follows:

[0112] ① Branching activation: PAMAM (G2~G6) with different molecular weight branches were prepared by taking the corresponding volume of branching concentrate according to the number of amino groups in 50 nmol, and then diluted to 0.2 mL with activation buffer at pH 7.2~8.0. 2-Iminithiothion hydrochloride (2IT) was selected as the branching activator, and a concentrate was prepared at a concentration of 5 mg / mL. According to the molar ratio of 2IT to amino groups of 0.5:1~2:1, the corresponding volume of 2IT concentrate (5 mg / mL) was added to the diluted branching solution, mixed well, and reacted at 25℃ for 1 h, followed by desalting treatment.

[0113] ②ALP activation: Measure 1.0 mg of ALP, desalt it first using a desalting column, and then adjust the concentration to 4 mg / mL using activation buffer. Mix the desalted ALP with SMCC at a molar ratio of 1:10, react at 25℃ for 1 h, and then perform desalting again.

[0114] ③ ALP polymerization: The activated and desalted branched chain and the activated and desalted ALP were mixed in a molar ratio of 1:2. After mixing, the mixture was reacted at 25°C for 1 hour. Then, 200 nmol of 2-MEA (2-mercaptoethylamine hydrochloride) was added to terminate the reaction for 30 minutes. Finally, 500 nmol of iodoacetamide was added to terminate the reaction for 30 minutes.

[0115] ④ Purification: The ALP polymer mixture was purified by size exclusion chromatography. The purified product was collected according to different peaks and stored at 2-8℃. The corresponding yield was calculated simultaneously. The yield was calculated as follows: Yield = (Amount of purified ALP polymers / Amount of theoretical ALP polymers) × 100%.

[0116] ⑤ Comparison of the stability of polymerized ALPs prepared with different molecular weight PAMAMs: The purified products (alkaline phosphatase polymers) of polymerized ALPs prepared with different molecular weight PAMAMs were prepared at 1 μg / mL and placed in a 37℃ oven for acceleration for 0, 3, 7, and 12 days. After acceleration, 10 μL of each product was added to the substrate to verify the stability of the alkaline phosphatase polymers before and after acceleration. Stability was reflected by calculating the increase in photometric value on day n of acceleration. The calculation method for the increase in photometric value on day n of acceleration was: Increase in photometric value on day n of acceleration = (Photometric value on day n of acceleration - Photometric value on day 0 / Photometric value on day 0) × 100%.

[0117] The results are shown in Table 1. In this embodiment, it was found that the molecular weight of PAMAM branches that is too high (e.g., G6 generation and above) or too low (e.g., G2 generation and below) will lead to a decrease in the yield of alkaline phosphatase polymers. Therefore, in this embodiment, G3, G4 and G5 PAMAM were initially selected as polymer backbones to prepare alkaline phosphatase polymers.

[0118] Table 1: Comparison of yields of alkaline phosphatase polymers prepared from PAMAMs of different molecular weights

[0119]

[0120] The results are shown in Table 2. Regardless of whether the acceleration was performed for 3, 7, or 12 days, the decrease in alkaline phosphatase polymers prepared by G2-G5 PAMAM after acceleration was controlled within 5%, meeting the stability requirements. However, the decrease in alkaline phosphatase polymers prepared by G6 PAMAM after 12 days of acceleration was close to 10%, which may be due to the larger size of the alkaline phosphatase polymers prepared by G6 PAMAM, leading to decreased stability.

[0121] Table 2: Comparison of stability of alkaline phosphatase polymers prepared from PAMAMs of different molecular weights

[0122]

[0123] 1.2 Optimization of Non-specific Adsorption of Alkaline Phosphatase Polymers

[0124] This embodiment uses five coupling agents with different chain lengths (i.e., SMCC, SM(PEG)2, SM(PEG)4, SM(PEG)8, and SM(PEG)). 12 Alkaline phosphatase polymers were prepared using [a specific method], and their yields and non-specific adsorption were compared. The specific preparation method is as follows:

[0125] ① Branching activation: Take a concentrated amount of G5 PAMAM branched solution according to the number of amino groups per 50 nmol, and dilute it to 0.2 mL with an activation buffer at pH 7.2-8.0. Select 2-iminothione hydrochloride (2IT) as the branching activator, and prepare a concentrated solution at a concentration of 5 mg / mL. Add the corresponding volume of 2IT concentrated solution (5 mg / mL) to the diluted branched solution according to a 2IT:amino group molar ratio of 1:1. Mix well and react at 25℃ for 1 h, then perform desalting treatment.

[0126] ②ALP activation: Measure 1.0 mg of ALP, desalt it first using a desalting column, and then adjust the concentration to 4 mg / mL using activation buffer. Activate the desalted ALP with SMCC, SM(PEG)2, SM(PEG)4, SM(PEG)8, and SM(PEG)3 respectively. 12Mix at a molar ratio of 1:10, react at 25°C for 1 hour after mixing, and then perform desalination treatment.

[0127] ③ ALP polymerization: The activated and desalted branched chains were mixed with the activated and desalted ALP at a molar ratio of 1:2. After mixing, the mixture was reacted at 25°C for 1 hour. 2-MEA was added to terminate the reaction for 30 minutes, followed by the addition of iodoacetamide to terminate the reaction for 30 minutes, with a final molar amount of 200 nmol.

[0128] ④ Purification: The ALP polymer mixture was purified by size exclusion chromatography. The purified product was collected and stored at 2-8℃, and the corresponding yield was calculated simultaneously.

[0129] ⑤ Comparison of non-specific adsorption of polymerized ALP activated by coupling agents of different chain lengths: Purified products of polymerized ALP activated by coupling agents of different chain lengths were prepared into working solutions of 1 μg / mL, and detected using antibody-coated magnetic beads. 30 μL of 1 mg / mL antibody-coated magnetic beads were incubated with 60 μL of polymerized ALP working solution for 10 min, and the results were repeated 10 times on a chemiluminescence instrument. The background differences were calculated and compared. Simultaneously, 10 μL of polymerized ALP working solution was added to the substrate to verify whether there were differences in photoluminescence values.

[0130] The results are shown in Table 3. This embodiment found that appropriately extending the chain length of the coupling agent can further improve the yield of alkaline phosphatase polymers, while excessively long coupling agent chains lead to a decrease in yield. Therefore, this embodiment preferably uses SM(PEG)2-8 as the coupling agent (activator) to prepare alkaline phosphatase polymers.

[0131] Table 3: Effect of coupling agents with different chain lengths on the yield of alkaline phosphatase polymers

[0132]

[0133] The results are shown in Table 4. In this embodiment, it was found that coupling agents containing PEG spacer arms (i.e., SM(PEG)) n This not only increases the yield of alkaline phosphatase polymers but also significantly reduces the non-specific adsorption of alkaline phosphatase polymers to magnetic beads while maintaining or even increasing alkaline phosphatase activity. SM(PEG) 12 While this reduces non-specific adsorption of magnetic beads, the excessively long chain length may conversely decrease alkaline phosphatase activity. Therefore, this embodiment selects SM(PEG) after comprehensive consideration. 2-8 (For example, SM(PEG)2, SM(PEG)4, SM(PEG)8) are used as coupling agents (activators) to prepare alkaline phosphatase polymers.

[0134] Table 4: Non-specific adsorption of magnetic beads by ALP polymerization mediated by coupling agents (activators) of different chain lengths

[0135]

[0136] Example 2

[0137] 2.1 Preparation of F(ab')2 fragment antibody:

[0138] ① Measure 1 mg of tau protein mouse monoclonal antibody, and replace the antibody with pH 2.5 citrate buffer through a desalting column to adjust the concentration to 4 mg / mL for later use;

[0139] ② Dissolve the pepsin powder in citrate buffer at pH 2.5 to a concentration of 4 mg / mL for later use;

[0140] ③ Mix the antibody and pepsin at a mass ratio of 5:1, and after mixing evenly, place the mixture in a 37°C oven for 2 hours.

[0141] ④ Adjust the pH of the reaction solution to 8.0 to terminate the enzymatic digestion reaction;

[0142] ⑤ Purification: The obtained F(ab')2 fragments were purified by filtration through molecular sieves, adjusted to the appropriate concentration, and stored at 2-8℃ for later use;

[0143] 2.2 Optimization of Alkaline Phosphatase Polymer Blocking

[0144] ① Branching activation: Take a concentrated amount of G5 PAMAM branched solution according to the number of amino groups per 50 nmol, and dilute it to 0.2 mL with an activation buffer at pH 7.2-8.0. Select 2-iminothione hydrochloride (2IT) as the branching activator, and prepare a concentrated solution at a concentration of 5 mg / mL. Add the corresponding volume of 2IT concentrated solution (5 mg / mL) to the diluted branched solution according to a 2IT:amino group molar ratio of 1:1. Mix well and react at 25℃ for 1 h, then perform desalting treatment.

[0145] ②ALP activation: Measure 1.0 mg of ALP, desalt it first using a desalting column, and then adjust the concentration to 4 mg / mL using activation buffer. Mix the desalted ALP with SM(PEG)4 at a molar ratio of 1:10, react at 25℃ for 1 h, and then perform desalting again.

[0146] ③ ALP polymerization: The activated and desalted branched chains were mixed with the activated and desalted ALP at a molar ratio of 1:2, and the mixture was reacted at 25℃ for 1 h. One group was terminated by adding 2-MEA with a final molar amount of 200 nmol for 30 min, followed by adding iodoacetamide with a final molar amount of 500 nmol for 30 min; the other group was terminated by adding cysteine ​​with a final molar amount of 200 nmol for 30 min, followed by adding N-(2-aminoethyl)maleimide with a final molar amount of 500 nmol for 30 min.

[0147] ④ Purification: The ALP polymer mixture was purified by size exclusion chromatography. The purified product was collected and the concentration was adjusted to 4 mg / mL. It was then stored at 2-8℃.

[0148] ⑤ F(ab')2 activation: Take 0.5 mg of the enzyme-digested F(ab')2 fragment antibody and adjust the concentration to 2 mg / mL. Select 2-iminothionane hydrochloride (2IT) as the activator and prepare a concentrated solution at a concentration of 5 mg / mL. Measure the appropriate volume of the 2IT concentrated solution according to a molar ratio of 2IT:F(ab')2 of 5:1 and add it to the above F(ab')2. Mix well and react at 25℃ for 1 h, then perform desalting treatment.

[0149] ⑥ Activation of polymerized ALP: Take 0.5 mg of polymerized ALP collected by different termination methods above, and add it for activation according to the molar ratio of polymerized ALP:SM(PEG)4 of 1:10. After mixing, react at 25℃ for 1 h, and then perform desalting treatment.

[0150] ⑦ Coupling of F(ab')2 with polymerized ALP: Measure the activated F(ab')2 and activated polymerized ALP at a mass ratio of 1:1, mix them well, and react at 25°C for 1 h.

[0151] ⑧ Purification: The F(ab')2 and polymerized ALP conjugate was purified by size exclusion chromatography. The purified product was collected and stored at 2-8℃. 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.

[0152] The results are shown in Table 5. In this embodiment, it was found that using cysteine ​​and N-(2-aminoethyl)maleimide as blocking agents can improve the coupling efficiency of F(ab')2 with polymerized ALP. The combination of cysteine ​​and N-(2-aminoethyl)maleimide as blocking agents is beneficial for subsequent reactivation coupling (i.e., coupling of F(ab')2 with polymerized ALP), and the yield of alkaline phosphatase polymer-antibody complex is higher.

[0153] Table 5: Effect of two different combinations of blocking agents on coupling efficiency

[0154]

[0155] 2.3 Preparation of F(ab')2 fragment antibody-polymerized ALP conjugate (alkaline phosphatase polymer-antibody complex)

[0156] ① Branching activation: Take the corresponding volume of G5 PAMAM branching concentrate according to the number of amino groups (50 nmol / mL), and then dilute it to 0.2 mL with activation buffer at pH 7.2-8.0. Select 2-iminothione hydrochloride (2IT) as the branching activator and prepare a concentrate at a concentration of 5 mg / mL. Measure the corresponding volume of 2IT concentrate (5 mg / mL) according to a 2IT:amino group molar ratio of 1:1 and add it to the diluted branching solution. Mix well and react at 25℃ for 1 h, then perform desalting treatment.

[0157] ②ALP activation: Measure 1.0 mg of ALP, desalt it first using a desalting column, and then adjust the concentration to 4 mg / mL using activation buffer. Mix the desalted ALP with SM(PEG)4 at a molar ratio of 1:10, react at 25℃ for 1 h, and then perform desalting again.

[0158] ③ ALP polymerization: The activated and desalted branched chains were mixed with the activated and desalted ALP at a molar ratio of 1:2, and the mixture was reacted at 25°C for 1 h. Cysteine ​​was added to terminate the reaction for 30 min with a final molar amount of 200 nmol, and then N-(2-aminoethyl)maleimide was added to terminate the reaction for 30 min with a final molar amount of 500 nmol.

[0159] ④ Purification: The ALP polymer mixture was purified by size exclusion chromatography. The purified product was collected and the concentration was adjusted to 4 mg / mL. It was then stored at 2-8℃.

[0160] ⑤ F(ab')2 activation: Take 0.5 mg of F(ab')2 fragment antibody from the enzyme-digested tau protein and adjust the concentration to 2 mg / mL. Select 2-iminothione hydrochloride (2IT) as the activator and prepare a concentrated solution at a concentration of 5 mg / mL. Add the appropriate volume of the 2IT concentrated solution to the above F(ab')2 at a molar ratio of 5:1 (2IT:F(ab')2), mix well, react at 25℃ for 1 h, and then perform desalting treatment.

[0161] ⑥ Activation of polymerized ALP: Take 0.5 mg of collected polymerized ALP and add it to activate it according to the molar ratio of polymerized ALP:SM(PEG)4 of 1:10. After mixing, react at 25℃ for 1 h and then desalt it.

[0162] ⑦ Coupling of F(ab')2 with polymerized ALP: Measure the activated F(ab')2 and activated polymerized ALP at a mass ratio of 1:1, mix them well, and react at 25°C for 1 h.

[0163] ⑧ Purification: The F(ab')2 and polymerized ALP conjugate were purified by size exclusion chromatography and the purified product was collected and stored at 2-8℃.

[0164] 2.4 Performance Validation of the Conjugate of F(ab')2 Fragment Antibody with Polymerized ALP

[0165] The F(ab')2 fragment antibodies used in this embodiment were all prepared by enzyme digestion of mouse monoclonal antibodies against tau protein. This embodiment mainly verifies performance by detecting two items: ptau181 and ptau217 (typical phosphorylated tau proteins). The reagents used in this embodiment are as follows:

[0166] Reagent 1: Component ①: 1 mg / mL magnetic beads coated with ptau181 specific antibody; Component ②: 0.5 μg / mL Tau-F(ab')2-polymerized ALP working solution;

[0167] Reagent 2: Component ①: 1 mg / mL magnetic beads coated with ptau217 specific antibody; Component ②: 0.5 μg / mL Tau-F(ab')2-polymerized ALP working solution;

[0168] Reagent 3: Component ①: 1 mg / mL magnetic beads coated with ptau181 specific antibody; Component ②: 0.5 μg / mL Tau-F(ab')2-ALP working solution;

[0169] Reagent 4: Component ①: 1 mg / mL magnetic beads coated with ptau217 specific antibody; Component ②: 0.5 μg / mL Tau-F(ab')2-ALP working solution;

[0170] Reagent 5: Component ①: 1 mg / mL magnetic beads coated with ptau181 specific antibody; Component ②: 0.5 μg / mL Tau-IgG-polymerized ALP working solution;

[0171] Reagent 6: Component ①: 1 mg / mL magnetic beads coated with ptau217 specific antibody; Component ②: 0.5 μg / mL Tau-IgG-polymerized ALP working solution;

[0172] Reaction mode: Add 50 μL of different concentrations of antigen working solution, add 30 μL of component ①, incubate for 10 min and then wash. After washing, add 60 μL of component ② and incubate for 10 min. After the reaction is complete, wash and then add substrate for photometric measurement.

[0173] 2.4.1 Sensitivity Verification

[0174] The four groups of reagents 1-4 were used to test the corresponding series of antigen working solutions. Each concentration point was tested twice. The average value of each point was calculated, and the corresponding signal-to-noise ratio (S / N) and background were compared.

[0175] 2.4.2 Precision Verification

[0176] High-concentration antigen ptau181 was added to normal human plasma samples to prepare low- and high-concentration samples at 1 pg / mL and 4 pg / mL, respectively. Similarly, high-concentration antigen ptau217 was added to normal human plasma samples to prepare low- and high-concentration samples at 0.4 pg / mL and 4 pg / mL, respectively. Each sample was tested 10 times, and the mean, standard deviation, and corresponding coefficient of variation (CV) were calculated. The coefficient of variation was calculated as: CV = (standard deviation / mean) × 100%.

[0177] 2.4.3 Verification of anti-interference capability

[0178] Five human anti-mouse antibody (HAMA) positive plasma samples and five rheumatoid factor (RF) positive plasma samples were selected and tested according to the reaction mode to compare the improvement in anti-interference ability.

[0179] 2.4.4 Thermal Accelerated Stability Verification

[0180] The reagent was placed in a 37℃ oven for 0, 7, and 12 days to accelerate its stability, and the stability before and after acceleration was compared.

[0181] The results are shown in Table 6. In this embodiment, it was found that the detection of ptau181 or ptau217 in blood showed that the sensitivity of the F(ab')2 fragment antibody and / or polymerized ALP conjugate prepared in this invention was significantly improved, the signal-to-noise ratio (S / N) was improved by about 4 to 5 times, and the background was relatively lower.

[0182] Table 6: Sensitivity Comparison

[0183]

[0184] The results are shown in Table 7. Compared with reagents 3 and 4, the intra-batch coefficient of variation of the F(ab')2 fragment antibody and polymerized ALP conjugate prepared in this invention (i.e., reagents 1 and 2) is smaller. This may be because the F(ab')2 fragment antibody and polymerized ALP conjugate prepared in this invention has better uniformity, thereby effectively improving the precision.

[0185] Table 7: Precision Comparison

[0186]

[0187] The results are shown in Table 8. In this embodiment, it was found that compared with conjugates prepared using IgG (reagents 5 and 6), the conjugates of F(ab')2 fragment antibody and polymerized ALP prepared in this invention (i.e., reagents 1 and 2) showed lower detection results for ptau181 and ptau217. They also showed significantly enhanced anti-interference ability when detecting HAMA and RF positive samples, which can reduce the cost of blocking agents.

[0188] Table 8:

[0189]

[0190] The results are shown in Tables 9 and 10. Compared with reagents 3 and 4, the conjugates of F(ab')2 fragment antibody and polymerized ALP prepared in this invention (i.e., reagents 1 and 2) showed lower thermal acceleration drops at 7 days and 12 days in the detection of ptau181 and ptau217, with the overall drop within 6.2%, meeting the stability requirements.

[0191] Table 9: Thermal Accelerated Stability Verification

[0192]

[0193] Table 10: Thermal Accelerated Stability Verification

[0194]

[0195] In summary, the alkaline phosphatase polymer-antibody complex prepared by this invention exhibits high yield, good stability, high coupling efficiency, and significantly improved sensitivity (4-5 times higher sensitivity in ptau181 and ptau217 tests). Furthermore, it has lower background, better precision, and stronger anti-interference capabilities (e.g., resistance to HAMA and RF interference). This reduces the cost of blocking agents while minimizing batch-to-batch variation and reducing non-specific adsorption, demonstrating significant advantages in detecting phosphorylated tau protein in blood samples.

[0196] The embodiments of the present invention have been described above, but the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and these forms are all within the protection scope of the present invention.

Claims

1. A method for improving the detection sensitivity of phosphorylated tau protein, characterized in that, The method includes the following steps: S101 Obtain a blood sample, wherein the blood sample contains phosphorylated tau protein; S102 Add the test reagent to the blood sample; S103 The concentration of phosphorylated tau protein in the blood sample was detected using a chemiluminescence method; The detection reagent comprises an alkaline phosphatase polymer-antibody complex. The alkaline phosphatase polymer includes a polymer backbone and an alkaline phosphatase linked 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. The coupling agent includes SM(PEG). n The molecular weight of PAMAM is selected from 3-30 kDa, and n is selected from integers from 2-8.

2. The method as described in claim 1, characterized in that, The antibody is a Fab fragment or an F(ab')2 fragment.

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

4. The method as described in claim 3, characterized in that, The first coupling agent and the second coupling agent comprise SM (PEG). n .

5. The method as described in 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 for phosphorylated tau protein, characterized in that, The detection reagent comprises an alkaline phosphatase polymer-antibody complex, wherein the alkaline phosphatase polymer comprises a polymer backbone and an alkaline phosphatase linked to the polymer backbone by a coupling agent, and an antibody is coupled to the alkaline phosphatase polymer. The polymer backbone is PAMAM, and the coupling agent includes SM(PEG). n The antibody specifically recognizes the phosphorylated tau protein; wherein the molecular weight of PAMAM is selected from 3-30 kDa, and n is selected from integers from 2-8.

7. The detection reagent as described in claim 6, characterized in that, The antibody is a Fab fragment or an F(ab')2 fragment.

8. The detection reagent as described in claim 6 or 7, characterized in that, The preparation method of the alkaline phosphatase polymer-antibody complex includes the following steps: S201 involves mixing and reacting the activated polymer backbone with alkaline phosphatase activated by the first coupling agent, then adding a blocking agent to terminate the reaction, thereby obtaining an alkaline phosphatase polymer. S202 The alkaline phosphatase polymer is activated with a second coupling agent to obtain an activated alkaline phosphatase polymer; S203 The activated antibody and the activated alkaline phosphatase polymer are mixed and reacted to obtain the alkaline phosphatase polymer-antibody complex; The blocking agent includes cysteine ​​and N-(2-aminoethyl)maleimide.

9. The detection reagent as described in 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 6-9.

11. SM(PEG) n The use of cysteine ​​and N-(2-aminoethyl)maleimide in the preparation of the detection reagent for testing phosphorylated tau protein as described in any one of claims 6-9, characterized in that, The molar ratio of cysteine ​​to N-(2-aminoethyl)maleimide is 1:10-4:5, where n is an integer from 2 to 8.

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