Phosphorylated Tau-231 protein chemiluminescence detection kit, antibody variable region and preparation method of antibody variable region

Abstract

The invention discloses a phosphorylated Tau-231 protein chemiluminescence detection kit, an antibody variable region and a preparation method of the antibody variable region, and relates to the technical field of in vitro detection.The kit comprises an AE-labeled tau antibody, a double magnetic particle reagent and a p-tau231 calibrator; wherein the double magnetic particle reagent comprises a p-tau231 antibody, the amino acid sequence of a light chain variable region of the p-tau231 antibody is shown as SEQ ID NO: 1, and the amino acid sequence of a heavy chain variable region of the p-tau231 antibody is shown as SEQ ID NO: 2. According to the technical scheme, the phosphorylated Tau-231 protein chemiluminiscence detection kit suitable for a full-automatic chemiluminiscence tester is established, and the kit is high in sensitivity and wide in linear range and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro detection, and in particular to a chemiluminescence detection kit for phosphorylated Tau-231 protein, an antibody variable region and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) is the most common type of dementia, accounting for 60% to 80% of all dementia cases. It is a neurodegenerative disease characterized by cognitive decline and memory loss. The typical pathological features of AD include Aβ plaques formed by β-amyloid protein deposition and neurofibrillary tangles caused by hyperphosphorylation of microtubule-associated tau protein. The methods for diagnosing AD mainly include cognitive function assessment, imaging examination, biomarker detection and gene detection.

[0003] Recent studies have shown that ultrasensitive detection techniques can identify AD-related pathological markers in the blood, such as p-tau231 (tau protein phosphorylated at position 231). As an early biomarker for AD, p-tau231 helps identify patients in the mild cognitive impairment (MCI) stage, and can also be used to monitor the progression of AD and evaluate the treatment effect. When used in combination with other biomarkers, it can improve the diagnostic accuracy of AD. There are various methods for detecting p-tau231, including enzyme linked immunosorbent assay (ELISA), immunoprecipitation mass spectrometry (IP-MS), liquid chromatography-mass spectrometry (LC-MS), single molecule array technology (Simoa) and chemiluminescence detection method. The classic and traditional ELISA method has the disadvantages of non-automation, requiring a large amount of samples, low sensitivity and poor precision; although IP-MS and LC-MS have high precision, they are expensive and cannot be widely used; Simoa has the advantages of high throughput and ultrasensitivity, but it is expensive and time-consuming for detection. The detection of p-tau231 based on chemiluminescence method has the characteristics of low variability between laboratories and batches, and is fully automated, with high reliability, short time consumption and suitability for clinical use. However, there is currently no p-tau231 assay kit for use with the MAGICL6000 fully automated chemiluminescence analyzer. These methods usually need to be used in combination with monoclonal antibodies that specifically recognize p-tau231, but the existing monoclonal antibodies on the market are few in number and low in sensitivity. Summary of the Invention

[0004] The technical solution of the present invention to solve the above technical problems is to provide a chemiluminescence detection kit for phosphorylated Tau-231 protein. The kit includes an acridinium ester (AE)-labeled tau antibody, a double magnetic particle reagent, and a p-tau231 calibrator. Among them, the double magnetic particle reagent includes a p-tau231 antibody, the amino acid sequence of its light chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of its heavy chain variable region is as shown in SEQ ID NO: 2.

[0005] Further, the double magnetic particle reagent includes: carboxyl magnetic particles coated with p-tau231 antibody and Tosyl magnetic particles coated with p-tau231 antibody, and they are mixed in a ratio of 1:1 - 3.

[0006] Further, the AE-labeled tau antibody is obtained by labeling the tau antibody with acridinium ester.

[0007] To solve the above technical problems, the present invention also proposes an antibody variable region for the p-tau231 antibody in the chemiluminescence detection kit for phosphorylated Tau-231 protein as described above. The CDR1 of the light chain of the p-tau231 antibody is QSVYNNNW, the CDR2 is SVS, and the CDR3 is LGDFDCSSADCGV; The CDR1 of the heavy chain of the p-tau231 antibody is GIDLSRNG, the CDR2 is IKGGGNA, and the CDR3 is ATSGSDY.

[0008] To solve the above technical problems, the present invention also proposes a preparation method for the double magnetic particle reagent for preparing the double magnetic particle reagent as described above, including the following steps: Pretreat and activate the carboxyl magnetic particles, then conjugate them with the p-tau231 antibody, and store them after blocking and washing. Pretreat the Tosyl magnetic particles, then conjugate them with the p-tau231 antibody, and store them after multiple blocking and washing. Mix the carboxyl magnetic particles coated with p-tau231 antibody and the Tosyl magnetic particles coated with p-tau231 antibody in a ratio of 1:1 - 3 to obtain the double magnetic particle reagent.

[0009] The technical solution of the present invention establishes a chemiluminescence detection kit for phosphorylated Tau-231 protein applicable to the MAGICL6000 fully automatic chemiluminescence analyzer. The kit has high sensitivity and a wide linear range, and has broad clinical application prospects. Description of the Drawings

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0011] Figure 1 It is the calibration result diagram of the carboxyl magnetic particle antibody, Tosyl magnetic particle antibody and double magnetic particle antibody of the present invention; Figure 2 It is the TEM result diagram of magnetic particles in different states of the present invention, where (a). Original carboxyl magnetic particles; (b). Coupled carboxyl magnetic particles; (c). Tosyl magnetic particles; (d). Coupled Tosyl magnetic particles; Figure 3 It is the DLS result diagram of magnetic particles in different states of the present invention, where A. Original carboxyl magnetic particles; B. Coupled carboxyl magnetic particles; C. Tosyl magnetic particles; D. Coupled Tosyl magnetic particles; Figure 4 It is the test result diagram of the detection ability of the reagent of the present invention. Detailed implementation manners

[0012] The present invention provides a chemiluminescence detection kit for phosphorylated Tau-231 protein, the variable region of the antibody and its preparation method, aiming to design a detection kit with high sensitivity and wide linear range.

[0013] Main experimental instruments and reagents: A. Experimental instruments; MAGICL6000 fully automatic chemiluminescence analyzer (JiDan Bio), digital display ordinary ultrasonic cleaner (XinZhi Bio), etc. B. Experimental reagents; The tau antibody and p-tau231 antigen are purchased from RenDuan Bio, the p-tau231 antibody is from Jilin University, bovine serum albumin is purchased from Roche, carboxyl magnetic particles and Tosyl magnetic particles are purchased from JiDan Bio, and acridinium ester is purchased from Shanghai MaiTuoWei, etc.

[0014] The following will illustrate the top block of the chemiluminescence detection kit for phosphorylated Tau-231 protein proposed by the present invention in specific embodiments: In the technical solution of this embodiment, as Figure 1As shown in the figure, a chemiluminescence detection kit for phosphorylated Tau-231 protein, the kit includes an AE-labeled tau antibody, a double magnetic particle reagent and a p-tau231 calibrator; wherein, the double magnetic particle reagent includes a p-tau231 antibody, the amino acid sequence of its light chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of its heavy chain variable region is as shown in SEQ ID NO: 2.

[0015] Further, the double magnetic particle reagent includes: Carboxyl magnetic particles coated with p-tau231 antibody and Tosyl magnetic particles coated with p-tau231 antibody.

[0016] Further, the AE-labeled tau antibody is obtained by labeling the tau antibody with acridinium ester.

[0017] Further, CDR1 of the light chain of the p-tau231 antibody is QSVYNNNW, CDR2 is SVS, and CDR3 is LGDFDCSSADCGV; CDR1 of the heavy chain of the p-tau231 antibody is GIDLSRNG, CDR2 is IKGGGNA, and CDR3 is ATSGSDY.

[0018] Example 1: A chemiluminescence detection kit for phosphorylated Tau-231 protein, the kit includes an AE-labeled tau antibody, a double magnetic particle reagent and a p-tau231 calibrator; wherein, the double magnetic particle reagent includes a p-tau231 antibody, the amino acid sequence of its light chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of its heavy chain variable region is as shown in SEQ ID NO: 2.

[0019] The amino acid sequence of the light chain variable region of the p-tau231 antibody is: AQVLTQTPSSVSAAVGGTVTINCQASQS VYNNNWLSWFQRKPGQPPKLLIHSVSTLYSGVSSQFKGSGSGTQFTLTIS DVQCDDAATYYCLGDFDCSSADCGVFGGGTEVVVR.

[0020] The full chain is AQVLTQTPSSVSAAVGGTVTINCQASQS VYNNNWLSWFQRKPGQPPKLLIHSVSTLYSGVSSQFKGSGSGTQFTLTIS DVQCDDAATYYCLGDFDCSSADCGVFGGGTEVVVR GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC; The amino acid sequence of the variable region of the heavy chain of the p-tau231 antibody is: QSLEESGGRLVTPGTPLTLTCTVSGIDLSRN GMNWVRQAPGKGLEWIAYIKGGGNAYYASWAKGRFTISKSSTTVDLKITT PTTEDTATYFCATSGSDYWGPGTLVTVSS。

[0021] The full chain is QSLEESGGRLVTPGTPLTLTCTVSGIDLSRN GMNWVRQAPGKGLEWIAYIKGGGNAYYASWAKGRFTISKSSTTVDLKITT PTTEDTATYFCATSGSDYWGPGTLVTVSS GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTV APSTCSKPTCP PPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPITHQDWLRGKEFKCKVHNKALPAPIEKTISKAR GQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK。 [[ID=?]]

[0022] The CDR1 of the light chain of the p-tau231 antibody is QSVYNNNW, the CDR2 is SVS, and the CDR3 is LGDFDCSSADCGV; The CDR1 of the heavy chain of the p-tau231 antibody is GIDLSRNG, the CDR2 is IKGGGNA, and the CDR3 is ATSGSDY.

[0023] 1. Preparation of tau antibody labeled with AE: Labeling: The tau antibody and acridinium ester were incubated in a water bath at 37 °C for 1 hour at a molar ratio of 1:25. Blocking: 5M Tris was added and incubated in a water bath at 37 °C for 30 minutes. Desalting and preservation: Pass through a desalting plate, collect the filtrate, add phosphate and glycerol, and then aliquot and store at -20 °C.

[0024] 2. Preparation of calibrators: The p-tau231 antigen was prepared into calibrators with seven concentrations of S0 (0 pg / mL), S1 (5 pg / mL), S2 (25 pg / mL), S3 (50 pg / mL), S4 (125 pg / mL), S5 (250 pg / mL), and S6 (500 pg / mL) using PBS containing 5% BSA as the calibrator diluent, and stored at 2 - 8 °C.

[0025] 3. Preparation of dual magnetic particle antibodies: (1) Preparation of carboxyl magnetic particle antibody: Pretreatment: The 1.5 - μm carboxyl magnetic particles were dispersed in 50 mM / L MES to a final concentration of 1% w / v, 5 mg / L of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was added, and the mixture was reacted at 37 °C at 220 rpm for 30 minutes. After magnetic separation, it was resuspended with MES again to obtain the activated carboxyl magnetic particles. Coupling: The p-tau231 antibody was diluted with MES to a final concentration of 0.02% w / v, slowly added to the carboxyl magnetic particles, and coupled at 37 °C at 220 rpm for 3 hours. After magnetic separation, it was resuspended and dispersed with 2-MorpholinoethanesulfonicAcid (MES). Blocking: 40 μL of 10% bovine serum albumin solution was added and blocked at 37 °C at 220 rpm for 90 minutes. Washing: After magnetic separation, the supernatant was discarded, and it was sonicated in a water bath for 5 minutes with 4 mL of 20 mM Tris containing 0.05% Tween20, and repeated 3 times. Preservation: Finally, the carboxyl magnetic particles coated with p-tau231 antibody were distributed in 50 mmol MES buffer.

[0026] (2) Preparation of Tosyl magnetic particle antibody: Pretreatment: Disperse 2-μm Tosyl magnetic particles in 100 mM Sodium Tetraborate (Na2B4O7) to a final concentration of 1% w / v; Coupling: Dilute the p-tau231 antibody with Na2B4O7 to a final concentration of 0.02% w / v, slowly add it to the carboxyl magnetic particles, couple at 37 °C and 220 rpm for 8 hours. After magnetic separation, resuspend and disperse with Na2B4O7; Blocking: Add 1 mL of 10% bovine serum albumin solution and block at 37 °C and 220 rpm for 8 hours; Washing: After magnetic separation, discard the supernatant, wash with 2 mL of 20 mM Tris containing 0.05% Tween20, and repeat the blocking and washing steps 3 times; Storage: Finally, distribute the carboxyl magnetic particles coated with the p-tau231 antibody in 50 mM MES buffer.

[0027] (3) Preparation of double magnetic particle antibody: Mix the carboxyl magnetic particles coated with the p-tau231 antibody and the Tosyl magnetic particles coated with the p-tau231 antibody in a ratio of 1:1 to obtain the double magnetic particle antibody.

[0028] To ensure the successful preparation of the magnetic particle antibody, dynamic light scattering and Zeta potential analysis were used to reveal the physical properties of the magnetic particle antibody; transmission electron microscopy can show the dispersion state of the magnetic particles before and after immunization.

[0029] Figure 2 The shown TEM images show that the original magnetic particles have a good morphology; after coupling the antibody, the magnetic particles remain stable, evenly dispersed, and are wrapped by a polymer shell, confirming the successful labeling of the antibody.

[0030] Figure 3 The shown particle size test shows that the carboxyl magnetic particles increase from 1546 nm to 2621 nm, and the Tosyl magnetic particles increase from 2147 nm to 2599 nm, indicating the successful coupling of the antibody. The Polymer Dispersion Index (PDI) is all less than 0.100, showing that all solutions are stably monodispersed, which helps to reduce the interaction between particles and improve the stability.

[0031] 4. Precision analysis of the kit in Example 1: Precision is one of the key indicators to measure the effectiveness of in vitro diagnostic reagents. Using the same batch of reagents, samples with medium and high levels of concentration were measured simultaneously, and the measurement was repeated 20 times. The mean, standard deviation, and coefficient of variation were calculated. As shown in Table 1 below, the within-batch precision of this kit is less than 7%.

[0032] Table 1 Precision Analysis:

[0033] 5. Accuracy Analysis of the Kit in Example 1: Normal saline and p-tau231 standard products with different concentrations were added to the self-made samples to prepare basic samples and recovery samples. Each sample was tested 3 times, and the recovery rate was calculated. The results are shown in Table 2. The recovery rates of the three samples were 92.00%, 107.00%, and 102.5% respectively, and the average recovery rate was 100.5%. The p-tau231 recovery rates of the three experimental samples were all between 90% and 110%, meeting the clinical requirements and proving that the reagent has good detection accuracy.

[0034] Table 2 Accuracy Analysis:

[0035] 6. Sensitivity Analysis of the Kit in Example 1: Generally, the following terms are used to describe the detection ability of the reagent, namely sensitivity: Limit of blank (LOB), Limit of detection (LOD), and Limit of quantitation (LOQ). Their specific meanings are as follows: LoB refers to the maximum value in the results of the blank sample series; LoD refers to the lowest concentration of the analyte that the method can detect; LoQ refers to the actual concentration that can reliably detect the analyte and the uncertainty of the detection result meets the established goals of the laboratory.

[0036] Theoretically, the relationship between the Limit of blank, Limit of detection, and Limit of quantitation is LoB < LoD ≤ LoQ.

[0037] Referring to EP17-A2 "Evaluation of the Detection Capability of Clinical Laboratory Measurement Procedures" issued by the American Society for Clinical Laboratory Standards, 5 blank samples were repeatedly tested 20 times to obtain a total of 100 detection results for normal distribution analysis. If the data conforms to the normal distribution, then LoB = μB + 1.645σB. The calculation method of LOD is similar to that of LOB.

[0038] 5 low-concentration samples were repeatedly tested 20 times under the same conditions using the same reagent, and the coefficient of variation was calculated. The total error was set at 30%. According to the coefficient of variation = 1 / 3 of the total error, the lowest detection concentration with a coefficient of variation ≤ 10% is LoQ.

[0039] The results are shown in Table 3: the blank limit values of the 5 samples were all less than 3 pg / mL, and the LoB was 3 pg / mL. The 100 test results conformed to the normal distribution, and the total standard deviation (σS) was calculated to be 0.23, resulting in a limit of detection LoD of 3.4 pg / mL.

[0040] Table 3 Determination results of blank samples:

[0041] Five low-concentration samples (4 pg / mL, 5 pg / mL, 6 pg / mL, 7 pg / mL, 8 pg / mL) were repeatedly tested 20 times each using the same reagent. The 100 test results conformed to the normal distribution, and the coefficient of variation of each sample was obtained. The results are as Figure 4 shown. The bar graph represents the test means of samples at different concentrations, and the line graph represents the coefficient of variation of the test results of samples at different concentrations. When the sample concentration ≥ 5 pg / mL, the coefficient of variation CV of the sample < 10% meets the clinical diagnostic reagent specification, that is, the limit of quantitation LoQ is 5 pg / mL.

[0042] 7. Linear analysis of the kit in Example 1: A high-concentration sample close to the upper limit of the linear range of 500 pg / mL and a low-concentration sample close to the lower limit of the linear range were used to prepare 8 dilution concentrations, and the reagent was tested respectively. The average value was taken for each of the 3 tests at each dilution concentration, and the deviation from the theoretical value and the linear correlation coefficient were calculated. The results are shown in Table 4, indicating that the kit has good linearity within the linear range of 5 - 500 pg / mL.

[0043] Table 4 Linear analysis

[0044] Example 2: A preparation method of a double magnetic particle reagent, comprising the following steps: (1) Preparation of carboxyl magnetic particle antibody: Pretreatment: The 1.5 μm carboxyl magnetic particles were dispersed in 50 mM / L MES to a final concentration of 1% w / v, 5 mg / L of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochlorid, EDC) was added, and the reaction was carried out at 37°C for 30 minutes at 220 revolutions per minute. After magnetic separation, it was resuspended with MES again to obtain the activated carboxyl magnetic particles; Coupling: The p-tau231 antibody was diluted with MES to a final concentration of 0.02% w / v, and slowly added to the carboxyl magnetic particles. The coupling was carried out at 37°C for 3 hours at 220 revolutions per minute. After magnetic separation, it was resuspended and dispersed with MES; Blocking: Add 40 μL of 10% bovine serum albumin solution and block at 37 °C for 90 minutes at 220 rpm. Washing: Separate the magnetic particles and discard the supernatant. Sonicate in 4 mL of 20 mM Tris containing 0.05% Tween 20 in a water bath for 5 min, and repeat 3 times. Storage: Finally, distribute the carboxyl magnetic particles coated with p-tau231 antibody in 50 mmol MES buffer.

[0045] (2) Preparation of Tosyl magnetic particle antibody: Pretreatment: Disperse 2 μm Tosyl magnetic particles in 100 mM Na2B4O7 to a final concentration of 1% w / v. Coupling: Dilute p-tau231 antibody with Na2B4O7 to a final concentration of 0.02% w / v, slowly add it to the carboxyl magnetic particles, couple at 37 °C for 8 hours at 220 rpm. After magnetic separation, resuspend and disperse with Na2B4O7. Blocking: Add 1 mL of 10% bovine serum albumin solution and block at 37 °C for 8 hours at 220 rpm. Washing: Separate the magnetic particles and discard the supernatant. Wash with 2 mL of 20 mM Tris containing 0.05% Tween 20, and repeat the blocking and washing steps 3 times. Storage: Finally, distribute the carboxyl magnetic particles coated with p-tau231 antibody in 50 mM MES buffer.

[0046] (3) Preparation of double magnetic particle antibody: Mix the carboxyl magnetic particles coated with p-tau231 antibody and Tosyl magnetic particles coated with p-tau231 antibody in a ratio of 1:1 to obtain the double magnetic particle antibody.

[0047] Example 3: An antibody variable region, which is used for the p-tau231 antibody in the chemiluminescent detection kit for phosphorylated Tau-231 protein as described in Example 1. The CDR1 of the light chain of the p-tau231 antibody is QSVYNNNW, the CDR2 is SVS, and the CDR3 is LGDFDCSSADCGV; The CDR1 of the heavy chain of the p-tau231 antibody is GIDLSRNG, the CDR2 is IKGGGNA, and the CDR3 is ATSGSDY.

[0048] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A chemiluminescence detection kit for phosphorylated Tau-231 protein, characterized in that, The kit includes an AE-labeled tau antibody, a dual magnetic particle reagent, and a p-tau231 calibrator; wherein, the dual magnetic particle reagent includes a p-tau231 antibody, the amino acid sequence of its light chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of its heavy chain variable region is as shown in SEQ ID NO:

2.

2. The chemiluminescence detection kit for phosphorylated Tau-231 protein according to claim 1, wherein The dual magnetic particle reagent includes: carboxyl magnetic particles coated with p-tau231 antibody and Tosyl magnetic particles coated with p-tau231 antibody, and they are mixed in a ratio of 1:1 - 3.

3. The chemiluminescent detection kit for phosphorylated Tau-231 protein according to claim 1, wherein The AE-labeled tau antibody is obtained by labeling the tau antibody with acridinium ester.

4. An antibody variable region, being the p-tau231 antibody in the chemiluminescent detection kit for phosphorylated Tau-231 protein as described in any one of claims 1 to 3, characterized in that, The CDR1 of the light chain of the p-tau231 antibody is QSVYNNNW, CDR2 is SVS, and CDR3 is LGDFDCSSADCGV; The CDR1 of the heavy chain of the p-tau231 antibody is GIDLSRNG, CDR2 is IKGGGNA, and CDR3 is ATSGSDY.

5. A preparation method of a double magnetic particle reagent for preparing the double magnetic particle reagent according to any one of claims 1 to 3, characterized in that, It includes the following steps: The carboxyl magnetic particles are pretreated and activated, then coupled with the p-tau231 antibody, and stored after blocking and washing. The Tosyl magnetic particles are pretreated, then coupled with the p-tau231 antibody, and stored after multiple times of blocking and washing. The carboxyl magnetic particles coated with p-tau231 antibody and the Tosyl magnetic particles coated with p-tau231 antibody are mixed in a ratio of 1:1 - 3 to obtain the dual magnetic particle reagent.

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