A buffer for reducing Aβ1-42 protein aggregation and its application

Abstract

The present invention discloses a buffer for reducing the aggregation of Aβ1‑42 protein and its application, belonging to the technical field of chemiluminescent immunoassay. The buffer contains 6-10‰ m / v inorganic salt, 2-5‰ m / v preservative, 0.8-1.5‰ v / v surfactant, 0.8-1.5% m / v BSA and 0.5-2% m / v sodium hexadecyl diphenyl ether disulfonate. Using the buffer of the present invention, when detecting Aβ1‑42 protein in a biological sample based on chemiluminescent immunoassay, the aggregation of Aβ1‑42 protein at the detection temperature can be significantly reduced, thereby improving the detection signal value, further improving the detection sensitivity and accuracy, and having very important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemiluminescent immunochemical analysis, and in particular, relates to a buffer for reducing Aβ1-42 protein aggregation and application thereof. Background Art

[0002] Amyloid β (Aβ or Abeta) is a series of protein peptides formed after cleavage of amyloid precursor protein (APP) by β- and γ-secretases, which are believed to be involved in the pathogenesis of Alzheimer's disease. The appearance of amyloid β plaques, extracellular protein deposits, in the brain is a major hallmark of Alzheimer's disease. These plaques are formed by the spontaneous aggregation of monomeric Aβ into soluble Aβ oligomers, which aggregate together to form insoluble fibrils.

[0003] Most Aβ peptides are 40 residues long (Aβ1-40), and a small number are 42 residues (Aβ1-42 protein). Because the two extra amino acids in Aβ1-42 protein are more likely to cause misfolding and aggregation, it is generally believed that Aβ1-42 protein has stronger neurotoxicity. There is evidence that increased plasma levels of Aβ1-42 protein are associated with Alzheimer's disease.

[0004] At present, the main methods for quantitative detection of Aβ1-42 protein in the existing technology include chemiluminescent immunoassay, fluorescent immunochromatography and single molecule array technology. Among them, fluorescent immunochromatography has low sensitivity and limited application scope; single molecule array technology requires the use of expensive equipment and reagents in practical applications, and the cost is relatively high; chemiluminescent immunoassay is widely used.

[0005] However, with current technology, when using chemiluminescence immunoassay to detect Aβ1-42 protein in plasma samples, the detection signal value is low, which in turn causes low detection sensitivity and poor repeatability of the test results, resulting in low credibility of the test results. Summary of the invention

[0006] In order to solve at least one of the above technical problems, the inventors conducted a series of explorations on detection reagents and detection conditions, and unexpectedly found that when sodium hexadecyl diphenyl ether disulfonate or ulva polysaccharide is added to the buffer, Aβ1-42 protein aggregation can be reduced, the detection signal value can be increased, and the detection sensitivity and accuracy can be improved, thereby completing the present invention. On this basis, the technical solution adopted by the present invention is as follows:

[0007] A first aspect of the present invention provides a buffer for reducing Aβ1-42 protein aggregation, wherein the buffer contains 6~10‰ m / v inorganic salt, 2~5‰ m / v preservative, 0.8~1.5‰ v / v surfactant, 0.8~1.5% m / v BSA and 0.5~2% m / v sodium hexadecyl diphenyl ether disulfonate.

[0008] In the practice of Aβ1-42 protein detection, the inventor unexpectedly discovered that at the detection temperature (generally around 37°C), the buffer solution that provides the reaction environment may cause the Aβ1-42 protein to fold and aggregate, thereby affecting the detection results. After a lot of exploration, the inventor obtained a method that can reduce the aggregation of Aβ1-42 protein at the detection temperature, thereby improving the sensitivity and accuracy of the detection results.

[0009] In theory, no matter which method (flow fluorescence, chemiluminescence, etc.) is used to detect the corresponding protein, the signal value obtained by the detection should be proportional to or linearly related to the protein concentration. If the linear relationship between the detection signal value and the concentration of samples with different concentrations is poor, the final test result will be inaccurate; further, if the linear relationship is good at high concentrations, but poor at low concentrations, it indicates that the detection sensitivity is not high, that is, it is not suitable for detecting low-concentration samples.

[0010] In some embodiments of the present invention, the sodium hexadecyl diphenyl oxide disulfonate can be replaced by ulva polysaccharide, and the effect is slightly reduced, but still significantly better than the buffer obtained by the prior art solutions and other substances.

[0011] In some embodiments of the present invention, the buffer is selected from one of PBS buffer, MOPS buffer, MES buffer, HEPES buffer and Tris-HCl buffer. In some preferred embodiments of the present invention, the buffer is Tris-HCl buffer. Further, the concentration of the buffer is 80 to 150 mmol / L. In some preferred embodiments of the present invention, the concentration of the buffer is 100 mmol / L.

[0012] In some embodiments of the present invention, the inorganic salt is NaCl, KCl, MgCl2 and / or ZnCl2. In some preferred embodiments of the present invention, the inorganic salt is NaCl, and more preferably, its concentration in the buffer is 9‰ m / v, i.e., the mass volume fraction is 0.9%.

[0013] In some embodiments of the present invention, the preservative is at least one of the KroVin series of preservatives and / or at least one of the ProClin series of preservatives.

[0014] In some specific embodiments of the present invention, the KroVin series preservatives include KroVin 100, KroVin 400, KroVin 500 and KroVin 750; the ProClin series preservatives include ProClin 50, ProClin 150, ProClin 200, ProClin 300, ProClin 950 and ProClin 5000.

[0015] In some preferred embodiments of the present invention, the preservative is ProClin 300, and more preferably, its concentration in the buffer is 3‰ m / v, i.e., the mass volume fraction is 0.3%.

[0016] In some embodiments of the present invention, the surfactant is selected from at least one of SDS, Triton X-100, Tween 80 and Tween 20. In some preferred embodiments of the present invention, the surfactant is Tween 20, and more preferably, its concentration in the buffer is 1‰ v / v, i.e., the volume fraction is 0.1%.

[0017] The second aspect of the present invention provides the use of any buffer described in the first aspect of the present invention in the preparation of a kit for detecting Aβ1-42 protein in a biological sample based on chemiluminescent immunoassay, wherein the buffer is used to provide a reaction environment for the binding of the Aβ1-42 protein and the corresponding antibodies, wherein the corresponding antibodies include anti-Aβ1-42 protein antibodies coupled to magnetic beads and / or anti-Aβ1-42 protein antibodies labeled with chemiluminescent substances.

[0018] In some embodiments of the present invention, the chemiluminescent substance is selected from one of the acridinium ester series, one of the luminol series, or one of the peroxy oxalate series.

[0019] The third aspect of the present invention provides a reaction solution, wherein the reaction solution is any buffer solution according to the first aspect of the present invention, containing Aβ1-42 protein and anti-Aβ1-42 protein antibody.

[0020] Beneficial Effects of the Invention

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] By using the buffer of the present invention, when detecting Aβ1-42 protein in a biological sample based on chemiluminescent immunoassay, the aggregation of Aβ1-42 protein at the detection temperature (about 37°C) can be reduced, thereby increasing the detection signal value, thereby improving the detection sensitivity and accuracy, and has very important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure shows the linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #0 and the concentration of Aβ1-42 protein in Example 1 of the present invention.

[0024] Figure 2 The figure shows the linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #1 and the concentration of Aβ1-42 protein in Example 2 of the present invention.

[0025] Figure 3 The figure shows the linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #2 and the concentration of Aβ1-42 protein in Example 3 of the present invention.

[0026] Figure 4 The figure shows the linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #3 and the concentration of Aβ1-42 protein in Example 4 of the present invention.

[0027] Figure 5 The linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #4 and the concentration of Aβ1-42 protein in Example 5 of the present invention is shown.

[0028] Figure 6 The figure shows the linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #5 and the concentration of Aβ1-42 protein in Example 6 of the present invention.

[0029] Figure 7 The figure shows the linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #6 and the concentration of Aβ1-42 protein in Example 7 of the present invention.

[0030] Figure 8 The linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #7 and the concentration of Aβ1-42 protein in Example 8 of the present invention is shown.

[0031] Fig. 9 The linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #8 and the concentration of Aβ1-42 protein in Example 9 of the present invention is shown.

[0032] Fig.10 The linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #9 and the concentration of Aβ1-42 protein in Example 10 of the present invention is shown.

[0033] Fig.11 The linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #10 and the concentration of Aβ1-42 protein in Example 11 of the present invention is shown.

[0034] Fig.12 The linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #11 and the concentration of Aβ1-42 protein in Example 12 of the present invention is shown.

[0035] Fig.13 The linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #12 and the concentration of Aβ1-42 protein in Example 13 of the present invention is shown.

[0036] Fig.14 The linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #13 and the concentration of Aβ1-42 protein in Example 14 of the present invention is shown.

[0037] Fig.15 The linear correlation between the detection signal value of Aβ1-42 protein in the buffer of formula #14 and the concentration of Aβ1-42 protein in Example 15 of the present invention is shown.

[0038] Fig.16 The linear relationship between the measured values ​​obtained by detecting Aβ1-42 protein samples using different methods in Example 16 of the present invention and the Aβ1-42 protein concentration is shown. DETAILED DESCRIPTION

[0039] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the tests and characterization methods used are all synchronized with the filing date of this application. Where applicable, the contents of any patent, patent application or disclosure involved in this application are fully incorporated herein by reference, and their equivalent patent families are also introduced as references, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0040] Numerical ranges in this application are approximate values, so unless otherwise specified, they may include values ​​outside the range. Numerical ranges include all values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For a range containing a value less than 1 or containing a fraction greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately regarded as 0.0001, 0.001, 0.01 or 0.1. For a range containing a single digit less than 10 (e.g., 1 to 5), 1 unit is generally regarded as 0.1. These are only specific examples of what is intended to be expressed, and all possible combinations of the values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0041] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, adjuvants or compounds. On the contrary, except for those necessary for operating performance, the term "essentially consisting of..." excludes any other components, steps or processes from the scope of any description of the term below. The term "consisting of..." does not include any components, steps or processes that are not specifically described or listed. Unless explicitly stated, the term "or" refers to the listed individual members or any combination thereof.

[0042] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0043] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0045] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0046] The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0047] Example 1 Detection of Aβ1-42 protein in plasma samples

[0048] 1. Samples to be tested

[0049] In the present invention, 15 plasma samples of patients with Alzheimer's disease were obtained, and the Aβ1-42 concentration values ​​were detected using the Aβ1-42 / Aβ1-40 / T-Tau / p-Tau-181 / α-synuclein combined detection kit (flow cytometry) produced by Jiangxi Saiji Biotechnology Co., Ltd.

[0050] 2. Buffer

[0051] The buffer formulation for the assay (formulation #0) is as follows:

[0052] Based on 1000 mL, it includes 12.14 g Tris, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 10 g BSA, and concentrated hydrochloric acid is used to adjust the pH to 7.2±0.1.

[0053] 3. Detection Methods

[0054] Mix 75 μL of Aβ1-42 protein antibody (abcam, catalog number ab205340) labeled with acridinium ester (Shanghai MacLean Biochemical Technology Co., Ltd., catalog number: D915609), 60 μL of blocking agent (scantibodies, catalog number: 3KC001), 50 μL of the sample to be tested, 50 μL of magnetic particles (Suzhou Zhiyi Microsphere Technology Co., Ltd.) coated with Aβ1-42 protein antibody (abcam, catalog number: ab201060), and 60 μL of buffer (formula #0), and incubate at 37°C for 20 min to form a sandwich structure.

[0055] Under the action of an external magnetic field, a cleaning solution was added to remove the unbound magnetic particles coated with Aβ1-42 protein antibodies and other substances, and then a substrate solution for the fully automatic immunoassay system was added, and the number of photons generated was proportional to the concentration of Aβ1-42 protein in the sample. The results are shown in Table 1.

[0056] Table 1 Detection results of Aβ1-42 protein in the buffer of formula #0

[0057]

[0058] The concentration value refers to the concentration of Aβ1-42 protein obtained by testing samples 1 to 15 using the Aβ1-42 / Aβ1-40 / T-Tau / p-Tau-181 / α-synuclein combined detection kit (flow cytometry) produced by Jiangxi Saiji Biotechnology Co., Ltd.

[0059] As can be seen from Table 1, the Aβ1-42 protein detection signal values ​​(RLU values) of all plasma samples were low, and the distinction between low and high values ​​was also poor. For example, the RLU value corresponding to 8.03 pg / mL was 2163, while the RLU value corresponding to 16.21 pg / mL was 2156.

[0060] In addition, the overall linear relationship is also very poor, such as Figure 1 As shown, R 2 Only 0.2121.

[0061] It can be seen that the detection result of Aβ1-42 protein using the buffer of formula #0 will be inaccurate.

[0062] The inventors analyzed and found that the reason for the above phenomenon may be that the Aβ1-42 protein is hydrophobic and will have a certain degree of aggregation; in addition, since the temperature used during detection is generally 37°C, at this temperature, the secondary structure of the Aβ1-42 protein tends to transform from α-helix (α-helix) to β-sheet (β-sheet), which accelerates the formation of β-sheet structure in the protein. The β-sheet will further promote the aggregation of Aβ1-42 protein to form insoluble fibers, which reduces the affinity between the antibody and the antigen, and ultimately causes the detection signal value to be low.

[0063] To verify the above reasons, the inventors took 4 plasma samples and tested Aβ1-42 protein at different temperatures (2~8℃, 18℃, 23℃ and 37℃) using the buffer of formula #0. The results are shown in Table 2.

[0064] Table 2 Detection results of Aβ1-42 protein in buffer of formula #0 at different temperatures

[0065]

[0066] As can be seen from Table 2, for the same sample, the signal values ​​are significantly different when tested at different temperatures, and as the temperature increases, the signal value shows a significant downward trend, indicating that temperature promotes the aggregation of Aβ1-42 protein, reducing the bound Aβ1-42 protein antibodies labeled with acridinium ester, thereby reducing the signal value.

[0067] To this end, the inventors optimized the buffer to reduce the aggregation of Aβ1-42 protein in a 37°C detection environment, increase the detection signal value, and thereby improve the detection sensitivity and accuracy.

[0068] Example 2 Optimization of the formulation of the detection buffer - replacing the Tris-HCl buffer system with the MES buffer system

[0069] The inventors replaced the Tris-HCl buffer system of Formula #0 in Example 1 with a MES buffer system to obtain a new buffer formula (Formula #1), as follows:

[0070] 1000 mL includes 40 g MES, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 10 g BSA, and 4 mol / L NaOH to adjust the pH to 6.0±0.1.

[0071] The 15 plasma samples were tested in the same manner as in Example 1. The results are shown in Table 3.

[0072] Table 3 Detection results of Aβ1-42 protein in the buffer of formula #1

[0073]

[0074] As can be seen from Table 3, the Aβ1-42 protein detection signal values ​​of all plasma samples were also low, and the distinction between low and high values ​​was also poor. For example, the RLU value corresponding to 8.03 pg / mL was 2289, while the RLU value corresponding to 19.52 pg / mL was 2178.

[0075] In addition, the overall linear relationship is also very poor, such as Figure 2 As shown, R 2 Only 0.0692.

[0076] It can be seen from this that the detection results of Aβ1-42 protein using the buffer of formula #1 will also be inaccurate.

[0077] Example 3 Optimization of the formulation of the detection buffer - replacing the Tris-HCl buffer system with the PBS buffer system

[0078] The inventors replaced the Tris-HCl buffer system of Formula #0 in Example 1 with a PBS buffer system to obtain a new buffer formula (Formula #2), as follows:

[0079] Based on 1000 mL, it includes 9.07 g Na2HPO4·12H2O, 1.15 g KH2PO4, 9 g NaCl, 3 g Proclin300, 1.0 mL Tween 20, 10 g BSA, and the pH is 7.2±0.1.

[0080] The 15 plasma samples were tested in the same manner as in Example 1. The results are shown in Table 4.

[0081] Table 4 Detection results of Aβ1-42 protein in the buffer of formula #2

[0082]

[0083] As can be seen from Table 4, the Aβ1-42 protein detection signal values ​​of all plasma samples were also low, and the distinction between low and high values ​​was also poor. For example, the RLU value corresponding to 8.03 pg / mL was 2178, while the RLU value corresponding to 17.56 pg / mL was 2146.

[0084] In addition, the overall linear relationship is also very poor, such as Figure 3 As shown, R 2 Only 0.3582.

[0085] It can be seen from this that the detection results of Aβ1-42 protein using the buffer of formula #2 will also be inaccurate.

[0086] Example 4 Optimization of the formulation of the detection buffer - adjusting the pH to about 6.0

[0087] The inventors adjusted the pH value of the Tris-HCl buffer system of Formula #0 in Example 1 from about 7.2 to about 6.0, and obtained a new buffer formula (Formula #3), as follows:

[0088] Based on 1000 mL, it includes 12.14 g Tris, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 10 g BSA, and concentrated hydrochloric acid is used to adjust the pH to 6.0±0.1.

[0089] The 15 plasma samples were tested in the same manner as in Example 1. The results are shown in Table 5.

[0090] Table 5 Aβ1-42 protein detection results in the buffer of formula #3

[0091]

[0092] As can be seen from Table 5, the Aβ1-42 protein detection signal values ​​of all plasma samples are also low, and the distinction between low values ​​and high values ​​is also poor. For example, the RLU value corresponding to 8.03 pg / mL is 2348, while the RLU value corresponding to 19.52 pg / mL is 2486.

[0093] In addition, the overall linear relationship is also very poor, such as Figure 4 As shown, R 2 Only 0.0006.

[0094] It can be seen from this that the detection results of Aβ1-42 protein using the buffer of formula #3 will also be inaccurate.

[0095] Example 5 Optimization of the formulation of the detection buffer - adjusting the pH to about 8.4

[0096] The inventor adjusted the pH value of the Tris-HCl buffer system of Formula #0 in Example 1 from about 7.2 to about 8.4 to obtain a new buffer formula (Formula #4), as follows:

[0097] Based on 1000 mL, it includes 12.14 g Tris, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 10 g BSA, and concentrated hydrochloric acid is used to adjust the pH to 8.4±0.1.

[0098] The 15 plasma samples were tested in the same manner as in Example 1. The results are shown in Table 6.

[0099] Table 6 Detection results of Aβ1-42 protein in the buffer of formula #4

[0100]

[0101] As can be seen from Table 6, the Aβ1-42 protein detection signal values ​​of all plasma samples are also low, and the distinction between low values ​​and high values ​​is also poor. For example, the RLU value corresponding to 8.03 pg / mL is 2048, while the RLU value corresponding to 17.86 pg / mL is 2098.

[0102] In addition, the overall linear relationship is also very poor, such as Figure 5 As shown, R 2 Only 0.0437.

[0103] It can be seen from this that the detection results of Aβ1-42 protein using the buffer of formula #4 will also be inaccurate.

[0104] Example 6 Optimization of the formulation of the detection buffer - addition of glycerol

[0105] The inventors added 10% glycerol to the formula #0 of Example 1 to obtain a new buffer formula (formula #5), as follows:

[0106] Based on 1000 mL, it includes 12.14 g Tris, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 10 g BSA, 100 mL glycerol, and concentrated hydrochloric acid to adjust the pH to 7.2±0.1.

[0107] The 15 plasma samples were tested in the same manner as in Example 1. The results are shown in Table 7.

[0108] Table 7 Aβ1-42 protein detection results in the buffer of formula #5

[0109]

[0110] As can be seen from Table 7, the Aβ1-42 protein detection signal values ​​of all plasma samples were also very low, only reaching the background level, and the distinction between low and high values ​​was also poor. For example, the RLU value corresponding to 8.03 pg / mL was 471, while the RLU value corresponding to 14.23 pg / mL was 475.

[0111] In addition, the overall linear relationship is also very poor, such as Figure 6 As shown, R 2 Only 0.0103.

[0112] It can be seen from this that the detection result of Aβ1-42 protein using the buffer of formula #5 will also be inaccurate.

[0113] Example 7 Optimization of the formulation of the detection buffer - addition of sucrose

[0114] The inventors added 5% sucrose to the formula #0 of Example 1 to obtain a new buffer formula (formula #6), as follows:

[0115] Based on 1000 mL, it includes 12.14 g Tris, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 10 g BSA, 50 g sucrose, and concentrated hydrochloric acid to adjust the pH to 7.2±0.1.

[0116] The 15 plasma samples were tested in the same manner as in Example 1. The results are shown in Table 8.

[0117] Table 8 Detection results of Aβ1-42 protein in the buffer of formula #6

[0118]

[0119] As can be seen from Table 8, the Aβ1-42 protein detection signal values ​​of all plasma samples are also very low, and the distinction between low values ​​and high values ​​is also poor. For example, the RLU value corresponding to 8.03 pg / mL is 2812, while the RLU value corresponding to 16.21 pg / mL is 2887.

[0120] In addition, the overall linear relationship is also very poor, such as Figure 7 As shown, R 2 Only 0.0003.

[0121] It can be seen from this that the detection results of Aβ1-42 protein using the buffer of formula #6 will also be inaccurate.

[0122] Example 8 Optimization of the formulation of the detection buffer - adjustment of the BSA concentration

[0123] The inventors adjusted the BSA concentration in Formula #0 of Example 1 from 1% to 5% to obtain a new buffer formula (Formula #7), as follows:

[0124] Based on 1000 mL, it includes 12.14 g Tris, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 50 g BSA, and concentrated hydrochloric acid is used to adjust the pH to 7.2±0.1.

[0125] The 15 plasma samples were tested in the same manner as in Example 1. The results are shown in Table 9.

[0126] Table 9 Aβ1-42 protein detection results in the buffer of formula #7

[0127]

[0128] As can be seen from Table 9, the Aβ1-42 protein detection signal values ​​of all plasma samples are also very low, and the distinction between low values ​​and high values ​​is also poor. For example, the RLU value corresponding to 8.03 pg / mL is 2186, while the RLU value corresponding to 20.76 pg / mL is 2148.

[0129] In addition, the overall linear relationship is also very poor, such as Figure 8 As shown, R 2 Only 0.1334.

[0130] It can be seen from this that the detection results of Aβ1-42 protein using the buffer of formula #7 will also be inaccurate.

[0131] Example 9 Optimization of the formulation of the detection buffer - adding dithiothreitol

[0132] The inventors added 1% dithiothreitol to the formula #0 of Example 1 to obtain a new buffer formula (formula #8), as follows:

[0133] Based on 1000 mL, it includes 12.14 g Tris, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 10 g BSA, 10 g dithiothreitol, and concentrated hydrochloric acid to adjust the pH to 7.2±0.1.

[0134] The 15 plasma samples were tested in the same manner as in Example 1. The results are shown in Table 10.

[0135] Table 10 Detection results of Aβ1-42 protein in the buffer of formula #8

[0136]

[0137] As can be seen from Table 10, the Aβ1-42 protein detection signal values ​​of all plasma samples are also very low, and the distinction between low values ​​and high values ​​is also poor. For example, the RLU value corresponding to 8.03 pg / mL is 2186, while the RLU value corresponding to 20.76 pg / mL is 2148.

[0138] In addition, the overall linear relationship is also very poor, such as Fig. 9 As shown, R 2 Only 0.1212.

[0139] It can be seen from this that the detection results of Aβ1-42 protein using the buffer of formula #8 will also be inaccurate.

[0140] Example 10 Optimization of the formulation of the detection buffer solution - adding sodium hexadecyl diphenyl ether disulfonate

[0141] The inventors added 1% sodium hexadecyl diphenyl ether disulfonate to the formula #0 of Example 1 to obtain a new buffer formula (formula #9), as follows:

[0142] Based on 1000 mL, it includes 12.14 g Tris, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 10 g BSA, 10 g sodium hexadecyl diphenyl ether disulfonate, and concentrated hydrochloric acid to adjust the pH to 7.2±0.1.

[0143] The 15 plasma samples were tested in the same manner as in Example 1. The results are shown in Table 11.

[0144] Table 11 Aβ1-42 protein detection results in the buffer of formula #9

[0145]

[0146] As shown in Table 11, the signal value of Aβ1-42 protein was significantly improved by using the buffer of formula #9 to detect the Aβ1-42 protein, and the signal value was proportional to the Aβ1-42 protein concentration in the plasma sample. The higher the Aβ1-42 protein concentration, the higher the signal value, and the correlation was very good. Fig.10 As shown, R 2 Up to 0.9949.

[0147] It can be seen that the sensitivity and accuracy of the detection results of Aβ1-42 protein using the buffer of formula #9 are very high.

[0148] Example 11 Optimization of the formulation of the detection buffer - adding benzalkonium bromide

[0149] The inventors added 1% benzalkonium bromide to the formula #0 of Example 1 to obtain a new formula of buffer (formula #10), as follows:

[0150] Based on 1000 mL, it includes 12.14 g Tris, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 10 g BSA, 10 g benzalkonium bromide, and concentrated hydrochloric acid to adjust the pH to 7.2±0.1.

[0151] The 15 plasma samples were tested in the same manner as in Example 1. The results are shown in Table 12.

[0152] Table 12 Detection results of Aβ1-42 protein in the buffer of formula #10

[0153]

[0154] As can be seen from Table 12, the Aβ1-42 protein detection signal values ​​of all plasma samples are also low, and the overall linear relationship is not ideal, such as Fig.11 As shown, R 2 Only 0.8784.

[0155] It can be seen from this that the detection results of Aβ1-42 protein using the buffer of formula #10 will also be inaccurate.

[0156] Example 12 Optimization of the formulation of the detection buffer - addition of lecithin

[0157] The inventors added 1% lecithin to the formula #0 of Example 1 to obtain a new buffer formula (formula #11), as follows:

[0158] Based on 1000 mL, it includes 12.14 g Tris, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 5 g BSA, 10 g lecithin, and concentrated hydrochloric acid to adjust the pH to 7.2±0.1.

[0159] The 15 plasma samples were tested in the same manner as in Example 1. The results are shown in Table 13.

[0160] Table 13 Detection results of Aβ1-42 protein binding in the buffer of formula #11

[0161]

[0162] As can be seen from Table 13, the Aβ1-42 protein detection signal values ​​of all plasma samples are also low, and the overall linear relationship is even less ideal, such as Fig.12 As shown, R 2 Only 0.4431.

[0163] It can be seen from this that the detection results of Aβ1-42 protein using the buffer of formula #11 will also be inaccurate.

[0164] Example 13 Optimization of the formulation of the detection buffer solution - adding fatty alcohol polyoxyethylene ether

[0165] The inventors added 1% fatty alcohol polyoxyethylene ether to the formula #0 of Example 1 to obtain a new buffer formula (formula #12), as follows:

[0166] Based on 1000 mL, it includes 12.14 g Tris, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 10 g BSA, 10 g fatty alcohol polyoxyethylene ether, and concentrated hydrochloric acid to adjust the pH to 7.2±0.1.

[0167] The same method as in Example 1 was used to test 25 plasma samples. The results are shown in Table 14.

[0168] Table 14 Detection results of Aβ1-42 protein binding in the buffer of formula #12

[0169]

[0170] As shown in Table 14, the Aβ1-42 protein detection signal values ​​of all plasma samples were significantly improved, but the linear relationship was not ideal, such as Fig.13 As shown, R 2 Only 0.786.

[0171] It can be seen from this that the detection results of Aβ1-42 protein using the buffer of formula #12 will also be inaccurate.

[0172] Example 14 Optimization of the formulation of the detection buffer - adding Ulva polysaccharide

[0173] The inventors added Ulva polysaccharide to the formulation #0 of Example 1 to obtain a new buffer formulation (formula #13), as follows:

[0174] Based on 1000 mL, it includes 12.14 g Tris, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 10 g BSA, 10 g Ulva polysaccharide, and the pH is adjusted to 7.2±0.1 with concentrated hydrochloric acid.

[0175] The 15 plasma samples were tested in the same manner as in Example 1. The results are shown in Table 15.

[0176] Table 15 Detection results of Aβ1-42 protein binding in the buffer of formula #13

[0177]

[0178] As shown in Table 15, the Aβ1-42 protein detection signal values ​​of all plasma samples were significantly improved, but the linear relationship was not ideal, such as Fig.14 As shown, R 2 It is 0.9039.

[0179] It can be seen that the accuracy of the detection results of Aβ1-42 protein was significantly improved by using the buffer of formula #13, but the effect was not as good as adding sodium hexadecyl diphenyl ether disulfonate.

[0180] Example 15 Optimization of the formulation of the detection buffer - adding brazilin

[0181] The inventors added 1% brazilin to the formula #0 of Example 1 to obtain a new buffer formula (formula #14), as follows:

[0182] Based on 1000 mL, it includes 12.14 g Tris, 9 g NaCl, 3 g Proclin 300, 1.0 mL Tween 20, 10 g BSA, 10 g brazilwood, and the pH is adjusted to 7.2±0.1 with concentrated hydrochloric acid.

[0183] The 15 plasma samples were tested in the same manner as in Example 1. The results are shown in Table 16.

[0184] Table 16 Detection results of Aβ1-42 protein binding in the buffer of formula #14

[0185]

[0186] As can be seen from Table 16, the Aβ1-42 protein detection signal values ​​of all plasma samples are relatively low, and the linear relationship is not ideal, such as Fig.15 As shown, R 2 It is 0.8315.

[0187] It can be seen that the accuracy of the detection results of Aβ1-42 protein using the buffer of formula #14 is improved, but it is not ideal.

[0188] Example 16 Detection of Aβ1-42 protein

[0189] In summary, the detection signal value of Aβ1-42 protein was high when the buffer solution of formula #9 was used to detect the Aβ1-42 protein, which can theoretically improve the detection sensitivity. To verify this, the inventors used the Aβ1-42 / Aβ1-40 / T-Tau / p-Tau-181 / α-synuclein combined detection kit (flow cytometry) produced by Jiangxi Saiji Biotechnology Co., Ltd. as a control to detect the Aβ1-42 protein sample.

[0190] Among them, Aβ1-42 protein samples were analyzed by using β-amyloid peptide (Aβ 42 ) pure solution standard material (China Institute of Metrology, GBW09875) was prepared, and the test results are shown in Table 17.

[0191] Table 17 Measured values ​​of Aβ1-42 protein detected by different methods

[0192]

[0193] Further, the concentration of Aβ1-42 protein in the Aβ1-42 protein sample was used as the horizontal axis, and a scatter plot was drawn using the values ​​measured by the method of Example 10 and the values ​​measured by the Jiangxi Secchi kit, and a linear curve was fitted, as shown in FIG. Fig.16 shown.

[0194] Depend on Fig.16 It can be seen that the linearity of the detection results of Aβ1-42 protein samples was poor (R 2 =0.8603). Further analysis revealed that when the Aβ1-42 protein concentration was lower than 3.9pg / mL, the measured value did not change significantly when detected using the Jiangxi Secchi test kit, indicating that the detection limit of the Jiangxi Secchi Biological Test Kit was higher than 3.9pg / mL. In addition, according to the manufacturer's instructions, the upper detection limit of the Jiangxi Secchi Biological Test Kit does not exceed 625pg / mL, resulting in the measured value of the 1000pg / mL sample being not much different from that of the 500pg / mL sample. On the whole, due to the high lower detection limit and low upper detection limit of the Jiangxi Secchi Biological Test Kit, the linear relationship between the MFI value and the Aβ1-42 protein concentration is poor in the range of 2.6~1000pg / mL of Aβ1-42 protein concentration. However, using the method of Example 10 for detection, the linear relationship between the measured value and the concentration is very good in the range of 2.6~1000pg / mL (R 2 =0.9983), indicating that the detection limit of the method in Example 10 can reach 2.6 pg / mL, and it also has a relatively accurate detection capability for high concentrations.

[0195] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of a buffer in the preparation of a kit for detecting Aβ1-42 protein in a biological sample based on chemiluminescent immunoassay, characterized in that: The buffer is used to provide a reaction environment for the Aβ1-42 protein and the corresponding antibody to combine to reduce the aggregation of the Aβ1-42 protein. The corresponding antibody includes an anti-Aβ1-42 protein antibody coupled to magnetic beads and / or an anti-Aβ1-42 protein antibody labeled with a chemiluminescent substance. The buffer contains 6~10‰ m / v inorganic salt, 2~5‰ m / v preservative, 0.8~1.5‰ v / v surfactant, 0.8~1.5% m / v BSA and 0.5~2% m / v sodium hexadecyl diphenyl ether disulfonate.

2. The use according to claim 1, characterized in that: The buffer is selected from one of PBS buffer, MOPS buffer, MES buffer, HEPES buffer and Tris-HCl buffer.

3. The use according to claim 2, characterized in that: The concentration of the buffer solution is 80-150 mmol / L.

4. The buffer according to claim 1, characterized in that The inorganic salt is NaCl, KCl, MgCl2 and / or ZnCl2.

5. The use according to claim 1, characterized in that: The preservative is at least one of the KroVin series preservatives and / or at least one of the ProClin series preservatives.

6. The use according to claim 5, characterized in that The KroVin series preservatives include KroVin 100, KroVin 400, KroVin 500 and KroVin 750; the ProClin series preservatives include ProClin 50, ProClin150, ProClin 200, ProClin 300, ProClin 950 and ProClin 5000.

7. The use according to claim 1, characterized in that The surfactant is selected from at least one of SDS, Triton X-100, Tween 80 and Tween 20.

8. The use according to claim 1, characterized in that: The chemiluminescent substance is selected from one of the acridinium ester series, one of the luminol series or one of the peroxy oxalate series.

Images