Brain-derived Tau protein single-molecule fluorescence immunoassay detection kit and use method thereof

Abstract

The invention discloses a brain-derived Tau protein single-molecule fluorescence immunoassay detection kit and a use method, the kit provided by the invention is composed of a BD-Tau magnetic bead reagent coated with a capture antibody, a BD-Tau calibrator, a BD-Tau quality control product, a biotinylated BD-Tau Detector reagent for detecting the antibody, a BD-Tau SBG reagent, an RGP reagent and a BD-Tau sample diluent, the magnetic bead concentration of the BD-Tau magnetic bead reagent is 0.04 mg / mL-0. 08mg / mL of carboxyl magnetic beads, the carboxyl magnetic beads are coated with Tau J5.H3 coated antibodies, the detection antibody in the BD-Tau Detector reagent of the biotinylated detection antibody is Tau12, the concentration of the detection antibody is 0.3 mu g / mL-0. 8mu g / mL, and the concentration of SBG in the BD-Tau SBG reagent is 40 pM-60 pM; the kit disclosed by the invention can be used by adopting any one of a one-step method, a two-step method or a three-step method to detect the content of the BD-Tau protein in a plasma sample, the detection limit is 0.050 pg / mL, and the detection range is 0.050 pg / mL-150 pg / mL; according to the invention, the specificity, accuracy and reliability of determination of the Alzheimer's disease are improved, and the problems of early diagnosis and early screening of the Alzheimer's disease are solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and relates to a single molecule fluorescence immunoassay kit for detecting brain-derived Tau protein and a method of using the same. Background Art

[0002] Alzheimer's disease is a neurodegenerative disease. Common detection methods include scale examination, PET-CT examination, and cerebrospinal fluid examination, which have the disadvantages of strong subjectivity, high cost, invasive examination, and low patient compliance. Plasma examination can effectively avoid these drawbacks. However, due to the existence of the blood-brain barrier, the concentrations of these biomarkers in plasma are lower, and for some items, they even reach the fg / mL level, which makes it impossible to detect them using traditional detection methods such as enzyme-linked immunosorbent assay and chemiluminescence immunoassay. Therefore, the research on diagnostic kits for Alzheimer's disease-related biomarkers has become one of the hot issues in the field of immunoassay.

[0003] Currently, a variety of Tau proteins (denoted as Tau) and a variety of phosphorylated Tau proteins (denoted as pTau) have been found to be biomarkers that are significantly correlated with Alzheimer's disease. There has been much research on kits for detecting pTau and Tau. However, the sources of both Tau and pTau are not limited to the brain but may also come from peripheral tissues such as the heart and muscle. Moreover, there are various types of Tau or pTau, and common subtypes include pTau-231, pTau-212, pTau-205, etc. It is impossible to effectively distinguish the tau formed in the brain and peripheral tissues, which reduces the specificity, accuracy, and reliability of the determination of Alzheimer's disease.

[0004] If a kit for detecting only brain-derived Tau, that is, brain-derived Tau protein (denoted as BD-Tau), can be designed, it will greatly improve the specificity, accuracy, and reliability of the determination of Alzheimer's disease, and has great significance for the accurate detection and determination of Alzheimer's disease. Summary of the Invention

[0005] The object of the present invention is to detect BD-Tau. Based on single molecule fluorescence immunoassay technology, a single molecule fluorescence immunoassay kit for brain-derived Tau protein is designed.

[0006] The technical solution adopted by the present invention provides a kit for detecting brain-derived Tau protein by single-molecule fluorescence immunoassay. The key lies in that the above-mentioned kit is composed of BD-Tau magnetic bead reagent coated with capture antibody, BD-Tau calibrator, BD-Tau quality control product, BD-Tau Detector reagent with biotinylated detection antibody, BD-Tau SBG reagent, RGP reagent and BD-Tau sample diluent; the magnetic beads of the above-mentioned BD-Tau magnetic bead reagent are carboxyl magnetic beads with a concentration of 0.04 mg / mL to 0.08 mg / mL, and the carboxyl magnetic beads are coated with Tau J5.H3 coating antibody; in the BD-Tau Detector reagent with biotinylated detection antibody, the detection antibody is Tau12, and the concentration of the detection antibody is 0.3 μg / mL to 0.8 μg / mL; the concentration of SBG in the above-mentioned BD-Tau SBG reagent is 40 pM to 60 pM.

[0007] Specifically, the preparation method of the above-mentioned BD-Tau sample diluent is as follows: Add 7.0 g to 9.0 g of tris(hydroxymethyl)aminomethane, 1.0 g to 1.6 g of tris(hydroxymethyl)aminomethane hydrochloride buffer salt, 6.0 g to 9.0 g of sodium chloride, 10 mL to 20 mL of non-ionic surfactant, 25 mL to 35 mL of blocker and 0.5 mL to 1.5 mL of preservative to 1500 mL of purified water, with a pH of 8.8 to 9.3, then add 2.0 g to 3.0 g of bovine serum albumin, stir, and filter through a membrane; the above-mentioned blocker is HBR-H1 blocker;

[0008] The preparation of the above-mentioned BD-Tau SBG reagent is to dilute the SBG concentrate with BD-Tau SBG diluent. The preparation method of the above-mentioned BD-Tau SBG diluent is as follows:

[0009] Add 100 mg to 110 mg of magnesium chloride to 1.0 mL to 2.0 mL of purified water, and vortex to mix evenly to obtain a magnesium chloride solution;

[0010] Add 1.0 g to 1.5 g of potassium dihydrogen phosphate, 10 g to 15 g of disodium hydrogen phosphate heptahydrate, 0.1 g to 0.5 g of potassium chloride, 6 g to 9 g of sodium chloride, 8 g to 15 g of sucrose, 0.8 mL to 2.5 mL of Tween-20, 0.3 mL to 1.0 mL of ProClin300 and 0.8 mL to 1.5 mL of the above-mentioned magnesium chloride solution to 1000 mL to 1500 mL of purified water, stir evenly, and control the pH value within the range of 7.2 to 7.6;

[0011] Then add 30 g to 40 g of bovine serum albumin and stir evenly.

[0012] Preferably, the diameter of the above-mentioned carboxyl magnetic beads is 2.4 μm to 3.0 μm, and the washed magnetic beads are activated with EDC solution for 20 min to 40 min; in the preparation process of the BD-Tau magnetic bead reagent, the coating antibody Tau J5.H3 needs to be subjected to liquid replacement and purification treatment before use, and is diluted with PBS buffer to a concentration of 0.1 mg / mL to 0.3 mg / mL to obtain the diluted coating antibody; the coating reaction is carried out in MES buffer. The specific process of the coating reaction is to add the activated magnetic beads to the MES buffer, vortex for 50 s to 80 s, remove the supernatant of the magnetic beads, add MES buffer again, vortex for 50 s to 80 s, ultrasonically treat for 8 s to 15 s, add the diluted coating antibody, vortex for 50 s to 80 s, and mix and react with a roller mixer for 1 hour to 3 hours. Use bovine serum albumin to block the coated magnetic beads, add magnetic bead preservation solution, and prepare the BD-Tau magnetic bead reagent.

[0013] Optimally, the preparation method of the above-mentioned magnetic bead preservation solution is as follows: Add 2.5 g to 3.5 g of tris(hydroxymethyl)aminomethane hydrochloride, 0.6 g to 1.0 g of tris(hydroxymethyl)aminomethane, 1 g to 2 g of sodium chloride, 8 mL to 12 mL of EDTA solution, 0.3 mL to 0.8 mL of Tween-20, and 0.1 mL to 0.5 mL of ProClin300 to 500 mL of purified water, stir at 1000 r / min to 1600 r / min for 3 min to 8 min, with the pH value in the range of 7.5 to 8.0, add 4.4 g to 5.3 g of bovine serum albumin, stir at 1000 r / min to 1600 r / min for 3 min to 8 min, and filter with a filter membrane.

[0014] Furthermore, the preparation process of the BD-Tau Detector reagent of the above-mentioned biotinylated detection antibody is as follows:

[0015] Add 8.0 μL to 12.0 μL of biotin labeling solution to 700 μL of detection antibody, vortex and mix, centrifuge, and incubate at room temperature for 50 min to 70 min. The above-mentioned detection antibody needs to be subjected to liquid replacement and purification treatment before use and is diluted with PBS buffer to 0.5 mg / mL to 2.0 mg / mL;

[0016] Add 50 μL to 90 μL of Tris-HCl solution to quench the reaction for 10 min to 20 min to obtain the antibody solution after biotinylation reaction;

[0017] Add the antibody after biotinylation reaction to the filter, add PBS buffer and centrifuge to obtain the recovered and concentrated antibody;

[0018] Prepare the BD-Tau Detector diluent, and dilute the recovered and concentrated antibody with the BD-Tau Detector diluent. After stirring, obtain the BD-Tau Detector reagent of the above-mentioned biotinylated detection antibody.

[0019] Furthermore, the preparation process of the above-mentioned biotin labeling solution is as follows: First, add dimethyl sulfoxide to NHS-PEG4-biotin to dissolve it, and vortex to mix evenly. Use 30 μL - 60 μL of dimethyl sulfoxide per milligram of NHS-PEG4-biotin to obtain a DMSO solution containing NHS-PEG4-biotin; then take the DMSO solution containing NHS-PEG4-biotin and add it to purified water, and vortex to mix evenly. The volume ratio of the DMSO solution containing NHS-PEG4-biotin to purified water is 1:2 - 4 to prepare the above-mentioned biotin labeling solution;

[0020] The preparation process of the above-mentioned BD-Tau Detector diluent is as follows: Add 0.3 g - 0.5 g of potassium dihydrogen phosphate, 3.0 g - 5.0 g of disodium hydrogen phosphate heptahydrate, 0.05 g - 0.10 g of potassium chloride, 2.5 g - 3.0 g of sodium chloride, 0.2 mL - 0.8 mL of non-ionic surfactant, 0.1 mL - 0.3 mL of preservative, and 3.0 mL - 4.0 mL of EDTA solution to 350 mL of purified water, and stir evenly; measure the pH value within the range of 7.2 - 7.6, and then add 6.0 g - 7.0 g of bovine serum albumin and stir evenly.

[0021] Preferably, the liquid change and purification treatment is carried out using an ultrafiltration tube or a dialysis bag.

[0022] The usage method of the brain-derived Tau protein single molecule fluorescence immunoassay kit. In the above-mentioned usage method, the above-mentioned single molecule fluorescence immunoassay kit is required to detect the content of BD-Tau protein in the plasma sample. The key lies in that the above-mentioned usage method is a one-step method, a two-step method, or a three-step method;

[0023] The above-mentioned one-step method is to incubate and wash the BD-Tau magnetic bead reagent coated with the capture antibody, the BD-Tau Detector reagent of the biotinylated detection antibody, the BD-Tau SBG reagent, and the sample to be analyzed in the above-mentioned kit together, collect the magnetic beads, mix them with the RGP reagent, and perform the test using single molecule fluorescence immunoassay technology;

[0024] In the above two-step method, the BD-Tau magnetic bead reagent coated with the capture antibody, the BD-Tau Detector reagent of the biotinylated detection antibody, and the sample to be analyzed in the above kit are incubated and washed together. The magnetic beads are collected, and then the BD-TauSBG reagent is added for a second incubation and washing. The magnetic beads are collected, mixed with the RGP reagent, and tested using single-molecule fluorescence immunoassay technology.

[0025] In the above three-step method, the analyte in the sample to be analyzed is captured by the BD-Tau magnetic bead reagent coated with the capture antibody first, and then the BD-Tau Detector reagent of the biotinylated detection antibody is added to form a captured analyte complex. Then, the BD-Tau SBG reagent is used to label the immunoanalyte. In the above three-step method, the magnetic beads are incubated and washed between each step.

[0026] Specifically, the above sample to be analyzed is obtained by diluting the plasma sample with the BD-Tau sample diluent by 0 to 4 times; the above incubation time is 5 min to 80 min, and the incubation temperature is 28 °C to 32 °C; in the above usage method, 1 magnetic bead is distributed in each micro-pit of the optical disc, and the volume of the micro-pit is 40 fL to 60 fL.

[0027] Preferably, in the above usage method, the BD-Tau calibrator is used for synchronous analysis to obtain the AEB value of the BD-Tau calibrator and fit the calibration curve; then, based on the AEB value of the sample to be analyzed or the AEB value of the BD-Tau quality control product and the calibration curve, the BD-Tau concentrations in the plasma sample and the quality control product are calculated; the correlation coefficient of the calibration curve > 0.9900, and B / A of the AEB value > 2.5.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention utilizes the difference in the protein structures of BD-Tau and peripheral-source Tau in splicing variants. There are six different-length Tau subtypes in the brain, and the Tau in peripheral tissues is distinguished from BD-Tau due to the insertion of exon 4a. By selecting suitable coating antibodies and detection antibodies, the present invention can specifically recognize the junction between exon 4 and exon 5 of BD-Tau, not only effectively distinguish peripheral-source Tau from BD-Tau, but also effectively identify different subtypes of BD-Tau.

[0030] Since BD-Tau only accounts for about one-fifth of the total Tau, and the effect of the blood-brain barrier causes the transfer of BD-Tau from the cerebrospinal fluid to the blood to be extremely small, which places extremely stringent requirements on the sensitivity and accuracy of the corresponding detection kit. The present invention optimizes the preparation method of the kit from multiple aspects, such as improving the purification method of antibodies, the selection of magnetic beads, the activation method of magnetic beads, specific antibodies, the reaction ratio of magnetic beads and antibodies, using special magnetic bead preservation liquid, optimizing the reaction ratio of biotin and antibodies, selecting suitable RGP reagents, and using BD-Tau calibrators and BD-Tau quality control products for traceability or assignment, etc., so that the detection limit of the kit reaches 0.050pg / mL, which greatly improves the sensitivity of the kit and can accurately detect extremely low levels of BD-Tau content in the sample to be tested. The sample amount required for the kit of the present invention is low, and only 25μL of sample is required for detection, and the plasma sample can be detected after 4-fold dilution.

[0031] In addition, the kit of the present invention can also be suitable for a variety of reaction methods, namely, one-step method, two-step method and three-step method. The reaction time of the one-step method is short, and the false results are higher than those of the two-step method and the three-step method; the reaction time of the three-step method is the longest, and the accuracy is higher than that of the one-step method and the two-step method. However, no matter which reaction method is used, the correlation coefficient of the calibration curve after multiple on-machine experimental verification is higher than 0.9900, and the B / A of the AEB value is higher than 2.5. This makes the method of using the kit of the present invention more selective.

[0032] In summary, the present invention realizes the detection of BD-Tau in plasma samples. The kit of the present invention can be used in conjunction with a fully automatic single-molecule fluorescence immunoassay analyzer. The present invention provides a complete on-machine detection reagent, and the test can be completed by only processing the sample or automatic dilution of the instrument. The operation is simple and the manual error is greatly reduced. The present invention applies high-sensitivity diagnostic technology to the detection of new low-abundance proteins, improves the specificity, accuracy and reliability of the determination of Alzheimer's disease, and solves the problem of early diagnosis and screening of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the linear verification result of the methodology verification of the present invention. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] For those not specified in the examples, conventional conditions can be followed; for reagents or instruments without specified manufacturers, they are all conventional products that can be obtained through commercial purchase.

[0036] The preparation of the reagents required in each of the following examples and comparative examples is described as follows:

[0037] 1) Preparation of PBS buffer: Add 0.04 g of potassium chloride, 0.044 g of potassium dihydrogen phosphate, 1.6 g of sodium chloride, and 0.46 g of disodium hydrogen phosphate heptahydrate to 200 mL of purified water, stir magnetically for 5 min, and control the pH value within the range of 7.2 ± 0.2;

[0038] 2) Preparation of washing buffer: Add 1 mL of Tween-20 to 99 mL of PBS buffer and stir magnetically for 5 min;

[0039] 3) Preparation of EDC solution: Take 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride out of the refrigerator and equilibrate at room temperature. Weigh 10 mg of EDC and add it to a 1.5 mL centrifuge tube, and then add 1 mL of 50 mM MES buffer, vortex and mix for 1 min;

[0040] 4) Prepare 0.1 M NaOH solution by conventional method, with water as the solvent;

[0041] 5) Prepare 50 mM MES buffer by conventional method, with water as the solvent, and control the pH at 6.1 - 6.3;

[0042] 6) Prepare 0.5 M EDTA solution by conventional method, with water as the solvent;

[0043] 7) Preparation method of Tris-HCl solution: Add 15.76 mg of Tris-HCl to a 2 mL centrifuge tube of purified water and vortex and mix.

[0044] In the present invention, the kits are prepared through Examples 1 to 3, and the prepared kits are all composed of BD-Tau magnetic bead reagent coated with capture antibody, BD-Tau calibrator, BD-Tau quality control product, BD-Tau Detector reagent with biotinylated detection antibody, BD-Tau SBG reagent, RGP reagent, and BD-Tau sample diluent.

[0045] Example 1

[0046] The specific preparation process of the kit is as follows:

[0047] S1. Preparation of BD-Tau magnetic bead reagent

[0048] In this embodiment, carboxyl magnetic beads with a diameter of 2.7 μm are selected, and the concentration is 35 mg / mL.

[0049] S1-1. Magnetic bead cleaning:

[0050] Take a 50 mL centrifuge tube, add 750 μL of magnetic beads, and remove the supernatant of the magnetic beads; add 15 mL of 0.1 M NaOH solution, and remove the supernatant of the magnetic beads again to clean the magnetic beads once;

[0051] Add 15 mL of washing buffer, remove the supernatant of the magnetic beads, and wash the magnetic beads three times;

[0052] Add 15 mL of 50 mM MES buffer, remove the supernatant of the magnetic beads, and wash the magnetic beads three times to obtain the washed magnetic beads.

[0053] S1-2. Magnetic bead activation:

[0054] Add 14.5 mL of 50 mM MES buffer and 500 μL of EDC solution to the centrifuge tube of the washed magnetic beads, vortex and mix for 60 s, and mix with a roller mixer for 30 min for activation;

[0055] After the activation is completed, remove the supernatant of the magnetic beads to obtain the activated magnetic beads.

[0056] S1-3. Magnetic bead coating:

[0057] S1-3-1. Preparation of coating antibody:

[0058] Take out the BD-Tau coating antibody from the refrigerator. The name of the BD-Tau coating antibody is Tau J5.H3, purchased from Bioventix Corporation. Equilibrate at room temperature. After complete melting, perform liquid exchange and purification treatment using a dialysis bag;

[0059] Dilute the BD-Tau coating antibody with PBS buffer to 0.2 mg / mL to obtain the diluted BD-Tau coating antibody.

[0060] S1-3-2. Coating reaction:

[0061] Add 15 mL of 50 mM MES buffer to the centrifuge tube of the activated magnetic beads, vortex and mix for 60 s, and remove the supernatant of the magnetic beads; add 12.5 mL of 50 mM MES buffer, vortex and mix for 60 s, and place it under the water surface of an ultrasonic cleaner for ultrasonic treatment for 10 s;

[0062] Add 1.5 mL of the diluted BD-Tau coating antibody, vortex for 60 s, mix with a roller mixer for 2 hours, and remove the supernatant of the magnetic beads to prepare the completely coated magnetic beads.

[0063] S1-4, Magnetic bead blocking:

[0064] Add 15 mL of washing buffer to the centrifuge tube containing the coated magnetic beads, vortex for 60 s, discard the supernatant of the magnetic beads, repeat the operation twice. Then add 15 mL of magnetic bead blocking solution, vortex for 60 s, and mix the blocking reaction on a roller mixer at room temperature for 45 min.

[0065] After the blocking is completed, discard the supernatant of the magnetic beads to obtain the blocked magnetic beads.

[0066] S1-5, Magnetic bead preservation

[0067] Preparation of magnetic bead preservation solution: Add 2.8 g of tris(hydroxymethyl)aminomethane hydrochloride, 0.8 g of tris(hydroxymethyl)aminomethane, 1.4 g of sodium chloride, 9.6 mL of 0.5 M EDTA solution, 0.5 mL of Tween-20, and 0.25 mL of ProClin300 to 500 mL of purified water, stir at 1200 r / min for 5 min, and the pH value of this example is 7.81; then add 4.9 g of bovine serum albumin, stir at 1200 r / min for 5 min, and filter with a 0.22 μm filter membrane.

[0068] Add 15 mL of washing buffer to the centrifuge tube containing the blocked magnetic beads, vortex for 60 s, discard the supernatant of the magnetic beads; add 420 mL of magnetic bead preservation solution, vortex for 60 s, and mix on a roller mixer for 30 min to obtain the BD-Tau magnetic bead reagent with a magnetic bead concentration of 0.0625 mg / mL.

[0069] S2, Preparation of BD-Tau Detector reagent:

[0070] S2-1, Preparation of detection antibody:

[0071] Take out the BD-Tau detection antibody from the refrigerator. The name of the BD-Tau detection antibody is Tau12, purchased from BioLegend. Equilibrate at room temperature until completely melted; perform buffer exchange and purification using a dialysis bag.

[0072] Dilute the BD-Tau detection antibody with PBS buffer to 1 mg / mL to obtain the diluted detection antibody.

[0073] S2-2, Preparation of biotin labeling solution:

[0074] Take out 2 mg of NHS-PEG4-biotin from the refrigerator, equilibrate at room temperature, add 100 μL of dimethyl sulfoxide to dissolve the biotin, and vortex to obtain a DMSO solution containing NHS-PEG4-biotin.

[0075] Take 9 μL of the DMSO solution containing NHS-PEG4-biotin, add 21 μL of purified water, and vortex to mix evenly to obtain the biotin labeling solution.

[0076] S2-3. Biotinylation reaction:

[0077] Add 9.5 μL of the biotin labeling solution to 700 μL of the diluted detection antibody, vortex to mix evenly, centrifuge briefly in a centrifuge, and incubate at room temperature for 60 min.

[0078] S2-4. Quenching:

[0079] Add 70 μL of 50 mM Tris-HCl solution to quench the reaction for 13 min to obtain the antibody solution after biotinylation reaction.

[0080] S2-5. Concentrated antibody recovery:

[0081] Add the antibody solution after biotinylation reaction to the filter, and add 720 μL of PBS buffer solution, then centrifuge;

[0082] Then add 1500 μL of PBS buffer solution to the filter and centrifuge, repeat the operation 4 times;

[0083] Flip the microcentrifuge tube to the top of the filter, assemble it by flipping, and centrifuge;

[0084] Add 200 μL of PBS buffer solution to the filter to wash the two layers of membranes in the filter, invert it in the microcentrifuge tube, and centrifuge to prepare the recovered and concentrated antibody.

[0085] S2-6. Dilution of concentrated antibody

[0086] Preparation method of BD-Tau Detector diluent: Add 0.38 g of potassium dihydrogen phosphate, 3.9 g of disodium hydrogen phosphate heptahydrate, 0.07 g of potassium chloride, 2.8 g of sodium chloride, 0.4 mL of Tween-20, 0.2 mL of ProClin300, and 3.5 mL of 0.5 M EDTA solution to 350 mL of purified water, stir for 20 min, measure the pH value within the range of 7.2 - 7.6. The pH value in this example is 7.35, add 6.9 g of bovine serum albumin, stir for 10 min, and filter with a 0.22 μm filter membrane;

[0087] Dilute the recovered and concentrated antibody to 0.5 μg / mL with the prepared BD-Tau Detector diluent, stir to obtain the BD-Tau Detector reagent.

[0088] S3. Preparation of BD-Tau sample diluent:

[0089] Take out the blocker HBR-H1 and bovine serum albumin from the refrigerator and equilibrate them at room temperature.

[0090] Add 8.2 g of tris(hydroxymethyl)aminomethane, 1.2 g of tris(hydroxymethyl)aminomethane hydrochloride, 7.5 g of sodium chloride, 28.5 mL of blocker HBR-H1, 15 mL of Triton X-100, and 0.8 mL of ProClin300 to 1500 mL of purified water, stir well, and control the pH value within the range of 8.8 - 9.3. The pH value in this implementation is 8.99.

[0091] Add 2.3 g of bovine serum albumin, stir well, and filter through a 0.22 μm filter membrane.

[0092] S4. Preparation of BD-Tau SBG reagent:

[0093] S4-1. Preparation of BD-Tau SBG diluent:

[0094] Add 1.1 mL of purified water, add the weighed 104.5 mg of magnesium chloride to 1.1 mL of purified water, and vortex to mix evenly.

[0095] Add 1.2 g of potassium dihydrogen phosphate, 12.2 g of disodium hydrogen phosphate heptahydrate, 0.22 g of potassium chloride, 8.7 g of sodium chloride, 11 g of sucrose, 1.1 mL of Tween-20, 0.55 mL of ProClin300, and 1.1 mL of magnesium chloride solution to 1100 mL of purified water, stir well, and the pH value is 7.45.

[0096] Add 33 g of bovine serum albumin to a beaker, stir well, and filter through a 0.22 μm filter membrane.

[0097] S4-2. Preparation of BD-Tau SBG reagent

[0098] Use the BD-Tau SBG diluent to dilute the SBG concentrate to a concentration of 50 pM of SBG, and stir well to obtain the BD-Tau SBG reagent.

[0099] S5. Preparation of RGP reagent:

[0100] S5-1. Preparation of RGP / DMSO reagent

[0101] Add 30 mg of resorufin-β-D-galactopyranoside to 1200 μL of dimethyl sulfoxide, and vortex to mix evenly to prepare the RGP / DMSO reagent.

[0102] S5-2. Preparation of RGP reagent

[0103] Mix 700 mL of PBS buffer and 1050 μL of RGP / DMSO solution, and stir to prepare the RGP reagent.

[0104] S6, BD-Tau calibration standard:

[0105] According to the tracing result of the BD-Tau recombinant antigen, dilute it to the target concentration, i.e., 150 pg / mL, with the BD-Tau sample diluent.

[0106] S7, BD-Tau quality control sample:

[0107] According to the labeled concentration of the BD-Tau recombinant antigen, dilute it to the target concentration with the BD-Tau sample diluent, and then assign values to the quality control sample using the assignment program. The assignment range is the target value ± 3SD.

[0108] The kit sample 1 is prepared through the above preparation process.

[0109] Example 2

[0110] The specific preparation process of the kit is as follows:

[0111] S1. Preparation of BD-Tau magnetic bead reagent

[0112] In this example, carboxyl magnetic beads with a diameter of 2.4 μm and a concentration of 30 mg / mL are selected.

[0113] S1-1. Magnetic bead cleaning:

[0114] Take a 50 mL centrifuge tube, add 720 μL of magnetic beads, and remove the supernatant of the magnetic beads; add 20 mL of 0.1 M NaOH solution, and remove the supernatant of the magnetic beads again to clean the magnetic beads once;

[0115] Add 20 mL of washing buffer, remove the supernatant of the magnetic beads, and clean the magnetic beads twice;

[0116] Add 20 mL of 50 mM MES buffer, remove the supernatant of the magnetic beads, and clean the magnetic beads four times to obtain the washed magnetic beads.

[0117] S1-2. Magnetic bead activation:

[0118] Add 16 mL of 50 mM MES buffer and 600 μL of EDC solution to the centrifuge tube with the washed magnetic beads, vortex and mix for 80 s, and mix with a roller mixer for 40 min for activation;

[0119] After the activation is completed, remove the supernatant of the magnetic beads to obtain the activated magnetic beads.

[0120] S1-3. Magnetic bead coating:

[0121] S1-3-1. Preparation of Coated Antibody:

[0122] Take out the BD-Tau coated antibody from the refrigerator. The name of the BD-Tau coated antibody is Tau J5.H3, purchased from Bioventix. Equilibrate at room temperature. After complete melting, perform liquid exchange and purification using an ultrafiltration tube;

[0123] Dilute the BD-Tau coated antibody with PBS buffer to 0.3 mg / mL to obtain the diluted BD-Tau coated antibody.

[0124] S1-3-2. Coating Reaction:

[0125] Add 20 mL of 50 mM MES buffer to the centrifuge tube of activated magnetic beads, vortex for 80 s, and remove the supernatant of the magnetic beads; add 15 mL of 50 mM MES buffer, vortex for 50 s, and place it under the water surface of an ultrasonic cleaner for ultrasonic treatment for 15 s;

[0126] Add 2.5 mL of the diluted BD-Tau coated antibody, vortex for 80 s, mix and react on a roller mixer for 3 hours, remove the supernatant of the magnetic beads, and prepare the completely coated magnetic beads.

[0127] S1-4. Magnetic Bead Blocking:

[0128] Add 20 mL of washing buffer to the centrifuge tube of the completely coated magnetic beads, vortex for 50 s, and remove the supernatant of the magnetic beads. Repeat the operation 2 times. Add 20 mL of magnetic bead blocking solution, vortex for 50 s, and perform blocking reaction on a roller mixer at room temperature for 50 min;

[0129] After blocking is completed, remove the supernatant of the magnetic beads to prepare the completely blocked magnetic beads.

[0130] S1-5. Magnetic Bead Preservation

[0131] Preparation of magnetic bead preservation solution: Add 3.5 g of tris(hydroxymethyl)aminomethane hydrochloride, 0.6 g of tris(hydroxymethyl)aminomethane, 2.0 g of sodium chloride, 12 mL of 0.5 M EDTA solution, 0.3 mL of Tween-20, and 0.5 mL of ProClin300 to 500 mL of purified water, stir at 1600 r / min for 3 min, and the pH value of this example is 8.00; add 5.3 g of bovine serum albumin, stir at 1600 r / min for 3 min, and filter with a 0.22 μm filter membrane.

[0132] Add 20 mL of washing buffer to the closed magnetic bead centrifuge tube, vortex for 50 s, and remove the supernatant of the magnetic beads; add 270 mL of magnetic bead storage solution, vortex for 50 s, and mix on a roller mixer for 20 min to prepare the BD-Tau magnetic bead reagent with a magnetic bead concentration of 0.080 mg / mL.

[0133] S2. Preparation of BD-Tau Detector reagent:

[0134] S2-1. Preparation of detection antibody:

[0135] Take out the BD-Tau detection antibody from the refrigerator. The name of the BD-Tau detection antibody is Tau12, purchased from BioLegend. Equilibrate at room temperature until completely melted; perform buffer exchange and purification using an ultrafiltration tube.

[0136] Dilute the BD-Tau detection antibody with PBS buffer to 2 mg / mL to obtain the diluted detection antibody.

[0137] S2-2. Preparation of biotin labeling solution:

[0138] Take out 2 mg of NHS-PEG4-biotin from the refrigerator, equilibrate at room temperature, add 120 μL of dimethyl sulfoxide to dissolve the biotin, and vortex to obtain a DMSO solution containing NHS-PEG4-biotin.

[0139] Take 9 μL of the DMSO solution containing NHS-PEG4-biotin, add 18 μL of purified water, and vortex to obtain the biotin labeling solution.

[0140] S2-3. Biotinylation reaction:

[0141] Add 12.0 μL of the biotin labeling solution to 700 μL of the diluted detection antibody, vortex, centrifuge briefly, and incubate at room temperature for 70 min.

[0142] S2-4. Quenching:

[0143] Add 90 μL of 50 mM Tris-HCl solution to quench the reaction for 10 min to obtain the antibody solution after biotinylation reaction.

[0144] S2-5. Concentration and antibody recovery:

[0145] Add the antibody solution after biotinylation reaction to the filter, and add 700 μL of PBS buffer, then centrifuge.

[0146] Then add 1200 μL of PBS buffer to the filter and centrifuge, repeat the operation 3 times.

[0147] Invert the microcentrifuge tube to the top of the filter, assemble by inversion, and centrifuge.

[0148] Add 180 μL of PBS buffer to the filter, wash the two layers of membranes in the filter, invert it in the microcentrifuge tube, and centrifuge to obtain the recovered and concentrated antibody.

[0149] S2-6. Dilution of the concentrated antibody

[0150] Preparation method of BD-Tau Detector diluent: Add 0.50 g of potassium dihydrogen phosphate, 3.0 g of disodium hydrogen phosphate heptahydrate, 0.10 g of potassium chloride, 2.5 g of sodium chloride, 0.8 mL of Tween-20, 0.3 mL of ProClin300, and 3.0 mL of 0.5 M EDTA solution to 350 mL of purified water, stir for 15 min. The pH value in this example is 7.60. Add 7.0 g of bovine serum albumin, stir for 15 min, and filter with a 0.22 μm filter membrane.

[0151] Dilute the recovered and concentrated antibody to 0.8 μg / mL with the prepared BD-Tau Detector diluent, stir to obtain the BD-Tau Detector reagent.

[0152] S3. Preparation of BD-Tau sample diluent:

[0153] Take out the blocker HBR-H1 and bovine serum albumin from the refrigerator and equilibrate at room temperature.

[0154] Add 9.0 g of tris(hydroxymethyl)aminomethane, 1.0 g of tris(hydroxymethyl)aminomethane hydrochloride, 9.0 g of sodium chloride, 35.0 mL of blocker HBR-H1, 10 mL of Triton X-100, and 1.5 mL of ProClin300 to 1500 mL of purified water, stir evenly. The pH value in this example is 9.3.

[0155] Add 3.0 g of bovine serum albumin, stir evenly, and filter with a 0.22 μm filter membrane.

[0156] S4. Preparation of BD-Tau SBG reagent:

[0157] S4-1. Preparation of BD-Tau SBG diluent:

[0158] Add the weighed 110 mg of magnesium chloride to 2.0 mL of purified water and vortex to mix evenly.

[0159] Dissolve 1.5 g of potassium dihydrogen phosphate, 10.0 g of disodium hydrogen phosphate heptahydrate, 0.50 g of potassium chloride, 9.0 g of sodium chloride, 15 g of sucrose, 0.8 mL of Tween-20, 1.0 mL of ProClin300, and 0.8 mL of magnesium chloride solution in 1000 mL of purified water, stir well, and the pH value is 7.59;

[0160] Add 40 g of bovine serum albumin to a beaker, stir well, and filter through a 0.22 μm filter membrane.

[0161] Preparation of S4-2 and BD-Tau SBG reagents

[0162] Dilute the SBG concentrate with BD-Tau SBG diluent to a concentration of 60 pM of SBG, and stir well to obtain the BD-Tau SBG reagent.

[0163] Preparation of S5, RGP reagent:

[0164] Preparation of S5-1, RGP / DMSO reagent

[0165] Add 26 mg of resorufin-β-D-galactopyranoside to 1000 μL of dimethyl sulfoxide, vortex to mix evenly, and prepare the RGP / DMSO reagent.

[0166] Preparation of S5-2, RGP reagent

[0167] Mix 700 mL of PBS buffer and 1150 μL of RGP / DMSO solution, stir, and prepare the RGP reagent.

[0168] S6, BD-Tau calibrator:

[0169] According to the result of tracing the BD-Tau recombinant antigen, dilute it to the target concentration with BD-Tau sample diluent, that is, 200 pg / mL.

[0170] S7, BD-Tau quality control product: The same as Example 1.

[0171] The kit sample 2 is prepared through the above preparation process.

[0172] Example 3

[0173] The specific preparation process of the kit is as follows:

[0174] Preparation of S1, BD-Tau magnetic bead reagent

[0175] In this example, carboxyl magnetic beads with a diameter of 3.0 μm and a concentration of 25 mg / mL are selected.

[0176] S1-1, Magnetic bead cleaning:

[0177] Take a 50 mL centrifuge tube, add 800 μL of magnetic beads, and remove the supernatant of the magnetic beads; add 18 mL of 0.1 M NaOH solution, and again remove the supernatant of the magnetic beads to wash the magnetic beads once.

[0178] Add 15 mL of washing buffer, remove the supernatant of the magnetic beads, and wash the magnetic beads three times.

[0179] Add 18 mL of 50 mM MES buffer, remove the supernatant of the magnetic beads, and wash the magnetic beads three times to obtain the washed magnetic beads.

[0180] S1-2. Magnetic bead activation:

[0181] Add 13.0 mL of 50 mM MES buffer and 400 μL of EDC solution to the centrifuge tube with the washed magnetic beads, vortex for 50 s, mix with a roller mixer, and activate for 20 min.

[0182] After activation, remove the supernatant of the magnetic beads to obtain the activated magnetic beads.

[0183] S1-3. Magnetic bead coating:

[0184] S1-3-1. Preparation of the coating antibody:

[0185] Take out the BD-Tau coating antibody from the refrigerator. The name of the BD-Tau coating antibody is Tau J5.H3, purchased from Bioventix Corporation. Equilibrate at room temperature. After complete melting, perform buffer exchange and purification using a dialysis bag.

[0186] Dilute the BD-Tau coating antibody with PBS buffer to 0.1 mg / mL to obtain the diluted BD-Tau coating antibody.

[0187] S1-3-2. Coating reaction:

[0188] Add 25 mL of 50 mM MES buffer to the centrifuge tube with the activated magnetic beads, vortex for 50 s, and remove the supernatant of the magnetic beads; add 10 mL of 50 mM MES buffer, vortex for 80 s, and place it under the water surface of an ultrasonic cleaner for ultrasonic treatment for 8 s.

[0189] Add 1.0 mL of the diluted BD-Tau coating antibody, vortex for 50 s, mix with a roller mixer for 1 hour, and remove the supernatant of the magnetic beads to prepare the completely coated magnetic beads.

[0190] S1-4. Magnetic bead blocking:

[0191] Add the centrifugal tube containing the coated magnetic beads to 18 mL of washing buffer, vortex for 80 s, remove the supernatant of the magnetic beads, repeat the operation 3 times, add 25 mL of magnetic bead blocking solution, vortex for 80 s, and mix the blocking reaction at room temperature on a roller mixer for 40 min;

[0192] After the blocking is completed, remove the supernatant of the magnetic beads to prepare the completely blocked magnetic beads.

[0193] S1-5. Preservation of magnetic beads

[0194] Preparation of magnetic bead preservation solution: Add 2.5 g of tris(hydroxymethyl)aminomethane hydrochloride, 1.0 g of tris(hydroxymethyl)aminomethane, 1.0 g of sodium chloride, 8 mL of 0.5 M EDTA solution, 0.8 mL of Tween-20, and 0.1 mL of ProClin300 to 500 mL of purified water, stir at 1000 r / min for 8 min, and the pH value of this example is 7.51; add 4.4 g of bovine serum albumin, stir at 1000 r / min for 8 min, and filter with a 0.22 μm filter membrane.

[0195] Add 20 mL of washing buffer to the centrifugal tube containing the completely blocked magnetic beads, vortex for 80 s, and remove the supernatant of the magnetic beads; add 500 mL of magnetic bead preservation solution, vortex for 80 s, place on a roller mixer and mix for 15 min to prepare the BD-Tau magnetic bead reagent with a magnetic bead concentration of 0.040 mg / mL.

[0196] S2. Preparation of BD-Tau Detector reagent:

[0197] S2-1. Preparation of detection antibody:

[0198] Take out the BD-Tau detection antibody from the refrigerator. The name of the BD-Tau detection antibody is Tau12, purchased from BioLegend, balance at room temperature and completely melt; perform liquid exchange and purification treatment using an ultrafiltration tube;

[0199] Dilute the BD-Tau detection antibody with PBS buffer to 1.5 mg / mL to obtain the diluted detection antibody.

[0200] S2-2. Preparation of biotin labeling solution:

[0201] Take out 2 mg of NHS-PEG4-biotin from the refrigerator, balance at room temperature, add 60 μL of dimethyl sulfoxide to dissolve the biotin, and vortex to obtain a DMSO solution containing NHS-PEG4-biotin;

[0202] Take 9 μL of the DMSO solution containing NHS-PEG4-biotin, add 36 μL of purified water, and vortex to obtain the biotin labeling solution.

[0203] S2-3, Biotinylation reaction:

[0204] Add 8.0 μL of biotin labeling solution to 700 μL of the diluted detection antibody, vortex mix, centrifuge briefly in a centrifuge, and incubate at room temperature for 50 min.

[0205] S2-4, Quenching:

[0206] Add 50 μL of 50 mM Tris-HCl solution to quench the reaction for 20 min to obtain the antibody solution after biotinylation reaction.

[0207] S2-5, Concentrated antibody recovery:

[0208] Add the antibody solution after biotinylation reaction to the filter, and add 680 μL of PBS buffer, then centrifuge;

[0209] Then add 1000 μL of PBS buffer to the filter and centrifuge, repeat the operation 4 times;

[0210] Flip the microcentrifuge tube to the top of the filter, assemble it by flipping, and centrifuge;

[0211] Add 220 μL of PBS buffer to the filter to wash the two layers of membranes in the filter, invert it in the microcentrifuge tube, and centrifuge to prepare the recovered and concentrated antibody.

[0212] S2-6, Dilution of concentrated antibody

[0213] Preparation method of BD-Tau Detector diluent: Add 0.30 g of potassium dihydrogen phosphate, 5.0 g of disodium hydrogen phosphate heptahydrate, 0.05 g of potassium chloride, 3.0 g of sodium chloride, 0.2 mL of Tween-20, 0.1 mL of ProClin300, and 4.0 mL of 0.5 M EDTA solution to 350 mL of purified water, stir for 25 min, the pH value in this example is 7.19, add 6.0 g of bovine serum albumin, stir for 20 min, and filter with a 0.22 μm filter membrane;

[0214] Dilute the recovered and concentrated antibody to 0.3 μg / mL with the prepared BD-Tau Detector diluent, stir to obtain the BD-Tau Detector reagent.

[0215] S3, Preparation of BD-Tau sample diluent:

[0216] Take out the blocker HBR-H1 and bovine serum albumin from the refrigerator and equilibrate them at room temperature;

[0217] Add 7.0 g of tris(hydroxymethyl)aminomethane, 1.6 g of tris(hydroxymethyl)aminomethane hydrochloride, 6.0 g of sodium chloride, 25.0 mL of blocker HBR-H1, 20 mL of Triton X-100, and 0.5 mL of ProClin300 to 1500 mL of purified water, stir evenly, and the pH value in this example is 8.81;

[0218] Add 2.0 g of bovine serum albumin, stir evenly, and filter with a 0.22 μm filter membrane.

[0219] S4. Preparation of BD-Tau SBG reagent:

[0220] S4-1. Preparation of BD-Tau SBG diluent:

[0221] Add the weighed 100 mg of magnesium chloride to 1.0 mL of purified water and mix well by vortexing;

[0222] Add 1.0 g of potassium dihydrogen phosphate, 15.0 g of disodium hydrogen phosphate heptahydrate, 0.10 g of potassium chloride, 6.0 g of sodium chloride, 8 g of sucrose, 2.5 mL of Tween-20, 0.3 mL of ProClin300, and 1.5 mL of magnesium chloride solution to 1500 mL of purified water, stir evenly, and the pH value is 7.21;

[0223] Add 30 g of bovine serum albumin to a beaker, stir evenly, and filter with a 0.22 μm filter membrane.

[0224] S4-2. Preparation of BD-Tau SBG reagent

[0225] Use the BD-Tau SBG diluent to dilute the SBG concentrate to a concentration of 40 pM of SBG, and stir evenly to prepare the BD-Tau SBG reagent.

[0226] S5. Preparation of RGP reagent:

[0227] S5-1. Preparation of RGP / DMSO reagent

[0228] Add 35 mg of resorcinol-β-D-galactopyranoside to 1300 μL of dimethyl sulfoxide and mix well by vortexing to prepare the RGP / DMSO reagent.

[0229] S5-2. Preparation of RGP reagent

[0230] Mix 700 mL of PBS buffer and 950 μL of RGP / DMSO solution, stir, and prepare the RGP reagent.

[0231] S6. BD-Tau calibrator: The same as in Example 1.

[0232] S7, BD-Tau Quality Control Sample: same as in Example 1.

[0233] The kit sample 3 was obtained through the above preparation process.

[0234] Comparative Example 1

[0235] The implementation method of this comparative example is the same as that of Example 1, except that in S1-3-1, Coating Antibody Preparation and S2-1, Detection Antibody Preparation, no liquid exchange and purification treatment were carried out, and the kit control product 1 was prepared.

[0236] Comparative Example 2

[0237] The implementation method of this comparative example is the same as that of Example 1, except that in S3, Preparation of BD-Tau Sample Diluent, no blocker was added, and the kit control product 2 was prepared.

[0238] Comparative Example 3

[0239] The implementation method of this comparative example is the same as that of Example 1, except that in S1-3-1, Coating Antibody Preparation process, the coating antibody used was the T-Tau monoclonal antibody 4F1 from Origene Company to prepare the kit control product 3.

[0240] Comparative Example 4

[0241] The implementation method of this comparative example is the same as that of Example 1, except that in S2-1, Detection Antibody Preparation process, the detection antibody used was the Tau antibody KC-240 from Absea Company to prepare the kit control product 4.

[0242] Experiment and Analysis

[0243] The kit samples prepared by the above examples were used for multiple on-machine tests, and the test instrument used was an automatic single-molecule fluorescence immunoassay analyzer.

[0244] In the study, the usage methods were divided into one-step method, two-step method and three-step method:

[0245] One-step method: The BD-Tau magnetic bead reagent coated with the capture antibody in the kit, the BD-Tau Detector reagent with biotinylated detection antibody, the BD-Tau SBG reagent and the sample to be analyzed were incubated and washed together, the magnetic beads were collected, mixed with the RGP reagent, and tested using single-molecule fluorescence immunoassay technology;

[0246] The two-step method involves incubating and washing the BD-Tau magnetic bead reagent coated with capture antibody, the BD-Tau Detector reagent with biotinylated detection antibody, and the sample to be analyzed in the kit, collecting the magnetic beads, adding the BD-Tau SBG reagent, performing a second incubation and washing, collecting the magnetic beads, mixing with the RGP reagent, and conducting the test using single molecule fluorescence immunoassay technology;

[0247] The three-step method is to capture the analyte in the sample to be analyzed with the BD-Tau magnetic bead reagent pre-coated with capture antibody, then add the BD-Tau Detector reagent with biotinylated detection antibody to form a captured analyte complex, and then label the immunoanalyte with the BD-Tau SBG reagent. In the three-step method described above, the magnetic beads are incubated and washed between each step.

[0248] Analysis Test One

[0249] The specific process using the two-step method is as follows:

[0250] Step 1: The reagent pipetting module of the analyzer aspirates 25 μL of the BD-Tau magnetic bead reagent and adds it to the reaction cup;

[0251] Step 2: The sample pipetting module of the analyzer uses a disposable conductive pipette tip to aspirate 6.25 μL of plasma sample and 18.75 μL of BD-Tau sample diluent and adds them to the reaction cup, that is, diluting the plasma sample 4 times to obtain the sample to be analyzed;

[0252] Step 3: The reagent pipetting module of the analyzer aspirates 20 μL of the BD-Tau Detector reagent and adds it to the reaction cup;

[0253] Step 4: Incubate at 30 °C for 60 min;

[0254] Step 5: The analyzer performs magnetic bead collection, washing, and collects the magnetic beads, discarding the supernatant;

[0255] Step 6: The reagent pipetting module of the analyzer aspirates 100 μL of the BD-Tau SBG reagent and adds it to the reaction cup, and resuspends the magnetic beads;

[0256] Step 7: Incubate at 30 °C for 5 min 15 s;

[0257] Step 8: The analyzer performs magnetic bead collection, washing, and collects the magnetic beads, discarding the supernatant;

[0258] Step 9: The sample pipetting module of the analyzer uses a disposable conductive pipette tip to aspirate 50 μL of the RGP reagent and adds it to the reaction cup, and resuspends the magnetic beads;

[0259] Step 10: The analyzer sample pipette module aspirates 20 μL of the mixed solution of the reacted magnetic beads and RGP reagent using a disposable conductive pipette tip and transfers it to the injection port of the optical disc. The RGP reagent flows into each micro-pit of the optical disc;

[0260] Step 11: The reacted magnetic beads are distributed into the micro-pits under the action of magnetic force, ensuring that there is only one magnetic bead in each micro-pit. The volume of each micro-pit is about 50 fL. Sealing oil is added, and the excess magnetic beads and RGP reagent are flushed out from the sample outlet;

[0261] Step 12: The magnetic beads containing β-galactosidase react with the RGP reagent in the micro-pits to produce resorufin. Resorufin produces emitted light at 585 nm under the action of excitation light at 577 nm. After taking pictures and making comparisons, data is obtained, statistical analysis of the data is carried out, and the value of each reaction, that is, the AEB value, is calculated according to the Poisson distribution principle;

[0262] Step 13: The instrument software fits a calibration curve through the calculated AEB value of the BD-Tau calibrator, and calculates the concentrations of BD-Tau in the plasma sample and the calibrator through the AEB value of the sample or calibrator and the calibration curve.

[0263] Analysis Test Two

[0264] The specific process of using the one-step method is as follows:

[0265] Step 1: The analyzer reagent pipetting module aspirates 25 μL of the BD-Tau magnetic bead reagent and adds it to the reaction cup;

[0266] Step 2: The analyzer sample pipette module aspirates 25 μL of the plasma sample using a disposable conductive pipette tip and adds it to the reaction cup, that is, the plasma sample is diluted 0 times to obtain the sample to be analyzed;

[0267] Step 3: The analyzer reagent pipetting module aspirates 20 μL of the BD-Tau Detector reagent and adds it to the reaction cup;

[0268] Step 4: The analyzer reagent pipetting module aspirates 100 μL of the BD-Tau SBG reagent and adds it to the reaction cup, and resuspends the magnetic beads;

[0269] Step 5: Incubate at 32 °C for 80 min;

[0270] The subsequent steps are the same as steps 8 to 13 of Analysis Test One, except that the volume of each micro-pit is about 40 fL.

[0271] Analysis Test Three

[0272] The specific process of using the three-step method is as follows:

[0273] Step 1: The analyzer reagent pipetting module aspirates 25 μL of BD-Tau magnetic bead reagent and adds it to the reaction cup;

[0274] Step 2: The analyzer sample pipetting module uses a disposable conductive pipette tip to aspirate 12.5 μL of plasma sample and 12.5 μL of BD-Tau sample diluent and adds them to the reaction cup, that is, diluting the plasma sample by 2 times to obtain the sample to be analyzed;

[0275] Step 3: Incubate at 28 °C for 20 min;

[0276] Step 4: The analyzer performs magnetic collection, washing, magnetic bead collection on the reaction cup, and discards the supernatant;

[0277] Step 5: The analyzer reagent pipetting module aspirates 20 μL of BD-Tau Detector reagent and adds it to the reaction cup;

[0278] Step 6: Incubate at 28 °C for 40 min;

[0279] Step 7: The analyzer performs magnetic collection, washing, magnetic bead collection on the reaction cup, and discards the supernatant;

[0280] Step 8: The analyzer reagent pipetting module aspirates 100 μL of BD-Tau SBG reagent and adds it to the reaction cup, and resuspends the magnetic beads;

[0281] Step 9: Incubate at 28 °C for 5 min;

[0282] The subsequent steps are the same as Steps 8 to 13 of Analysis Experiment 1, with the difference being that the volume of each micro-pit is about 60 fL.

[0283] Effect analysis

[0284] I. Using Analysis Experiment 1 to Analysis Experiment 3 respectively, test Kit Samples 1 to 3 and Kit Reference Standards 1 to 4, and the results of the correlation coefficient of the calibration curve and the B / A result of the AEB value are shown in Table 1.

[0285] Table 1: Summary of test results of kit samples and kit reference standards under different analysis experiments

[0286]

[0287] It can be seen from the results in Table 1 that the correlation coefficient of the calibration curve of the kit sample prepared by the present invention > 0.9900, and the B / A of the AEB value > 2.5. Without purifying the coating antibody and the detection antibody, substances such as protective proteins and preservatives in the commercial antibody will interfere with the reaction substances; if the blocking agent is not added to the BD-Tau sample diluent, it may cause non-specific binding and reduce the accuracy of the kit.

[0288] II. Using the method of Analytical Test 1, perform a specificity test on Kit Sample 1 to determine the anti-interference ability of the kit of the present invention:

[0289] Prepare different anti-interference ability test samples:

[0290] Anti-interference ability test sample 1: Add 2 μL of pTau-231 antigen sample with a known concentration of 200 pg / mL and 2 μL of pTau-212 antigen sample with a known concentration of 200 pg / mL to 200 μL of human plasma sample with a known BD-Tau concentration of 1.28 pg / mL to prepare Anti-interference ability test sample 1;

[0291] Anti-interference ability test sample 2: Add 2 μL of BD-Tau antigen sample with a known concentration of 1200 pg / mL, 2 μL of pTau-231 antigen sample with a known concentration of 1200 pg / mL, and 2 μL of pTau-205 antigen sample with a known concentration of 800 pg / mL to 200 μL of human plasma sample with a known BD-Tau concentration of 20.16 pg / mL to prepare Anti-interference ability test sample 2;

[0292] Anti-interference ability test sample 3: Add 2 μL of BD-Tau antigen sample with a known concentration of 3500 pg / mL, 2 μL of pTau-212 antigen sample with a known concentration of 3500 pg / mL, and 2 μL of pTau-205 antigen sample with a known concentration of 3500 pg / mL to 200 μL of human plasma sample with a known BD-Tau concentration of 87.67 pg / mL to prepare Anti-interference ability test sample 3;

[0293] Use Kit Sample 1 to test BD-Tau in Anti-interference ability test samples 1 to 3 respectively. Each test sample is tested 3 times repeatedly, and the average value is taken as the test result. The results are shown in Table 2.

[0294] Table 2: Summary table of test results of different anti-interference ability test samples

[0295]

[0296] As can be seen from the results in Table 2, the coating antibody and the detection antibody used in the present invention are specific. The coating antibody used in the present invention is a monoclonal antibody that specifically recognizes BD-Tau, which can ensure specific binding to an epitope of the target analyte; the detection antibody used in the present invention is an antibody specific for Tau protein, which can bind to another antigenic epitope of the analyte. Under the combined action of the two, the kit of the present invention can specifically recognize the junction between exon 4 and exon 5 of BD-Tau, and can not only effectively distinguish peripheral Tau from BD-Tau including different subtypes.

[0297] Methodology verification

[0298] Prepare the verification kit samples for methodology verification in the manner of Example 1, and perform verification by the two-step method. The verification process is as follows:

[0299] 1. Precision

[0300] 1.1 Intra-assay precision

[0301] Using the kit of the same batch number, for the samples to be analyzed in the concentration ranges of 8.0 pg / mL to 12.0 pg / mL (sample to be analyzed 1) and 80.0 pg / mL to 120 pg / mL (sample to be analyzed 2), repeat the measurement 10 times respectively, and calculate the average value of the measurement results. Standard deviation (SD) and coefficient of variation (CV), and the results are shown in Table 3.

[0302] Table 3: Intra-assay precision verification results

[0303]

[0304] 1.2 Inter-assay precision

[0305] Using kits of 3 different batch numbers, for the samples to be analyzed in the ranges of 8.0 pg / mL to 12.0 pg / mL (sample to be analyzed 1) and 80.0 pg / mL to 120 pg / mL (sample to be analyzed 2), repeat the measurement 10 times respectively, and calculate the average value of the 30 measurement results. Standard deviation (SD) and coefficient of variation (CV), and the results are shown in Table 4.

[0306] Table 4: Intra-assay precision verification results

[0307]

[0308]

[0309] 2. Detection limit

[0310] Five low-value samples to be analyzed with a concentration approximate to 0.050 pg / mL were detected, each detected 5 times. The detection results were sorted by size, and the number of detection results lower than the blank limit (0.020 pg / mL) should be less than or equal to 3. The results are shown in Table 5.

[0311] Table 5: Detection Limit Verification Results

[0312]

[0313] 3. Linearity

[0314] High-value samples (150 pg / mL) close to the upper limit of the linear range and low-value samples (0.050 pg / mL) close to the lower limit were diluted to at least 5 concentrations in a certain proportion. Each concentration was tested at least 2 times repeatedly, and the average value was calculated. The average value of the measured concentration and the dilution ratio were linearly fitted, and the linear correlation coefficient r was calculated. The results are shown in Table 6 and Appendix Figure 1 .

[0315] Table 6: Linearity Verification Results

[0316]

[0317]

[0318] 4. Accuracy

[0319] 10 μL of BD-Tau antigen sample with a known concentration of 800 pg / mL was added to 200 μL of human plasma sample with a known BD-Tau concentration of 2.57 pg / mL. The detected concentration after adding the BD-Tau antigen sample to the human plasma sample was 41.3 pg / mL, and the recovery rate detected and calculated was 102%.

[0320] From the above verification, it can be seen that the detection limit of the kit prepared by the present invention for BD-Tau is 0.050 pg / mL, the detection range is 0.050 pg / mL to 150 pg / mL, and the recovery rate is 102% within the range of 85% to 115%.

[0321] It can be seen that the present invention can improve the specificity, accuracy and reliability of the determination of Alzheimer's disease, and solve the problems of early diagnosis and early screening of Alzheimer's disease.

Claims

1. A single-molecule fluorescent immunoassay kit for detecting brain-derived Tau protein, characterized in that: The kit consists of a capture antibody-coated BD-Tau magnetic bead reagent, a BD-Tau calibrator, a BD-Tau quality control product, a biotinylated detection antibody BD-TauDetector reagent, a BD-Tau SBG reagent, an RGP reagent, and a BD-Tau sample diluent; the magnetic beads of the BD-Tau magnetic bead reagent are carboxyl magnetic beads with a concentration of 0.04 mg / mL to 0.08 mg / mL, and the carboxyl magnetic beads are coated with Tau J5.H3 coated antibodies; the detection antibody in the biotinylated detection antibody BD-Tau Detector reagent is Tau12, and the detection antibody concentration is 0.3 μg / mL to 0.8 μg / mL; the concentration of SBG in the BD-Tau SBG reagent is 40 pM to 60 pM.

2. The single-molecule fluorescent immunoassay kit for detecting brain-derived Tau protein according to claim 1, characterized in that: The preparation method of the BD-Tau sample diluent is as follows: 7.0g to 9.0g of trihydroxyaminomethane, 1.0g to 1.6g of trihydroxyaminomethane hydrochloride buffer salt, 6.0g to 9.0g of sodium chloride, 10mL to 20mL of non-ionic surfactant, 25mL to 35mL of blocking agent and 0.5mL to 1.5mL of preservative are added to 1500mL of purified water, the pH value is 8.8 to 9.3, and then 2.0g to 3.0g of bovine serum albumin is added, stirred, and filtered through a filter membrane; the blocking agent is HBR-H1 blocking agent; The preparation of the BD-Tau SBG reagent is to dilute the SBG concentrate using the BD-Tau SBG diluent, and the preparation method of the BD-Tau SBG diluent is: Add 100 mg to 110 mg of magnesium chloride to 1.0 mL to 2.0 mL of purified water and vortex mix to obtain a magnesium chloride solution; Add 1.0 g to 1.5 g of potassium dihydrogen phosphate, 10 g to 15 g of disodium hydrogen phosphate heptahydrate, 0.1 g to 0.5 g of potassium chloride, 6 g to 9 g of sodium chloride, 8 g to 15 g of sucrose, 0.8 mL to 2.5 mL of Tween-20, 0.3 mL to 1.0 mL of ProClin300, and 0.8 mL to 1.5 mL of the magnesium chloride solution to 1000 mL to 1500 mL of purified water, stir evenly, and control the pH value within the range of 7.2 to 7.6; Add 30g to 40g of bovine serum albumin and stir evenly.

3. The single-molecule fluorescent immunoassay kit for detecting brain-derived Tau protein according to claim 1, characterized in that: The diameter of the carboxyl magnetic beads is 2.4 μm to 3.0 μm, and the cleaned magnetic beads are activated with an EDC solution for 20 min to 40 min. In the preparation process of the BD-Tau magnetic bead reagent, the coated antibody Tau J5.H3 needs to be purified by changing the solution before use, and is diluted with a PBS buffer to a concentration of 0.1 mg / mL to 0.3 mg / mL to obtain a diluted coated antibody. The coating reaction is carried out in an MES buffer, and the specific process of the coating reaction is to add the activated magnetic beads to the MES buffer, vortex mix for 50s to 80s, remove the magnetic bead supernatant, add the MES buffer, vortex mix for 50s to 80s, ultrasonically treat for 8s to 15s, add the diluted coated antibody, vortex treat for 50s to 80s, mix and react with a roller mixer for 1 hour to 3 hours, block the coated magnetic beads with bovine serum albumin, add the magnetic bead preservation solution, and prepare the BD-Tau magnetic bead reagent.

4. The single-molecule fluorescent immunoassay kit for detecting brain-derived Tau protein according to claim 3, characterized in that: The preparation method of the magnetic bead preservation solution is as follows: 2.5g to 3.5g of tris(hydroxymethyl)aminomethane hydrochloride), 0.6g to 1.0g of tris(hydroxymethyl)aminomethane, 1g to 2g of sodium chloride, 8mL to 12mL of EDTA solution, 0.3mL to 0.8mL of Tween-20 and 0.1mL to 0.5mL of ProClin300 are added to 500mL of purified water, stirred at 1000r / min to 1600r / min for 3min to 8min, the pH value is within the range of 7.5 to 8.0, 4.4g to 5.3g of bovine serum albumin is added, stirred at 1000r / min to 1600r / min for 3min to 8min, and filtered with a filter membrane.

5. The single-molecule fluorescent immunoassay kit for detecting brain-derived Tau protein according to claim 1, characterized in that: The preparation process of the BD-Tau Detector reagent of the biotinylated detection antibody is as follows: Add 8.0 μL to 12.0 μL of biotin labeling solution to 700 μL of detection antibody, vortex mix, centrifuge, and incubate at room temperature for 50 min to 70 min. The detection antibody needs to be purified by changing the solution and diluted to 0.5 mg / mL to 2.0 mg / mL with PBS buffer before use; Add 50 μL to 90 μL of Tris-HCl solution, quench the reaction for 10 min to 20 min, and obtain an antibody solution after biotinylation reaction; The biotinylated antibody was added to the filter, and PBS buffer was added and centrifuged to obtain the recovered concentrated antibody; Prepare BD-Tau Detector diluent, and use the BD-Tau Detector diluent to dilute the recovered concentrated antibody, and stir to obtain the BD-Tau Detector reagent containing the biotinylated detection antibody.

6. The single-molecule fluorescent immunoassay kit for detecting brain-derived Tau protein according to claim 5, characterized in that: The preparation process of the biotin labeling solution is as follows: firstly add dimethyl sulfoxide to dissolve NHS-PEG4-biotin, vortex mix, use 30μL to 60μL dimethyl sulfoxide per mg of NHS-PEG4-biotin, and prepare a DMSO solution containing NHS-PEG4-biotin; then take the DMSO solution containing NHS-PEG4-biotin, add it to purified water, vortex mix, the volume ratio of the DMSO solution containing NHS-PEG4-biotin to purified water is 1:2 to 4, and the biotin labeling solution is prepared; The preparation process of the BD-Tau Detector diluent is as follows: add 0.3g to 0.5g of potassium dihydrogen phosphate, 3.0g to 5.0g of disodium hydrogen phosphate heptahydrate, 0.05g to 0.10g of potassium chloride, 2.5g to 3.0g of sodium chloride, 0.2mL to 0.8mL of non-ionic surfactant, 0.1mL to 0.3mL of preservative and 3.0mL to 4.0mL of EDTA solution to 350mL of purified water, and stir evenly; when the pH value is determined to be in the range of 7.2 to 7.6, add 6.0g to 7.0g of bovine serum albumin and stir evenly.

7. The single-molecule fluorescent immunoassay kit for detecting brain-derived Tau protein according to any one of claims 3 or 5, characterized in that: Fluid exchange and purification are performed using ultrafiltration tubes or dialysis bags.

8. A method for using a single-molecule fluorescent immunoassay kit for brain-derived Tau protein, wherein the method requires the use of a single-molecule fluorescent immunoassay kit for brain-derived Tau protein as described in any one of claims 1 to 7 to detect the content of BD-Tau protein in a plasma sample, characterized in that: The method of use is any one of a one-step method, a two-step method or a three-step method; The one-step method is to incubate and wash the capture antibody-coated BD-Tau magnetic bead reagent, the biotinylated detection antibody BD-Tau Detector reagent, the BD-Tau SBG reagent and the sample to be analyzed in the kit, collect the magnetic beads, mix them with the RGP reagent, and use the single molecule fluorescent immunoassay technique for testing; The two-step method is to incubate and wash the capture antibody-coated BD-Tau magnetic bead reagent, the biotinylated detection antibody BD-Tau Detector reagent and the sample to be analyzed together, collect the magnetic beads, then add the BD-Tau SBG reagent, perform a second incubation and washing, collect the magnetic beads, mix with the RGP reagent, and use the single molecule fluorescent immunoassay technique for testing; The three-step method is to first capture the analyte in the sample to be analyzed with a BD-Tau magnetic bead reagent coated with a capture antibody, then add a BD-Tau Detector reagent with a biotinylated detection antibody to form a captured analytical complex, and then label the immune analyte with a BD-Tau SBG reagent. In the three-step method, the magnetic beads will be incubated and washed between each step.

9. The method for using the brain-derived Tau protein single molecule fluorescent immunoassay detection kit according to claim 8, characterized in that: The sample to be analyzed is obtained by adding BD-Tau sample diluent to a plasma sample and diluting it 0 to 4 times; the incubation time is 5 minutes to 80 minutes, and the incubation temperature is 28°C to 32°C; in the method of use, one magnetic bead is distributed in each micropit of the optical disc, and the volume of the micropit is 40fL to 60fL.

10. The method for using the single-molecule fluorescent immunoassay kit for brain-derived Tau protein according to claim 8, characterized in that: The method of use requires the use of BD-Tau calibrators for synchronous analysis to obtain the AEB value of the BD-Tau calibrators and fit the calibration curve; then the BD-Tau concentration in the plasma sample and the quality control product is calculated using the AEB value of the sample to be analyzed or the AEB value of the BD-Tau quality control product and the calibration curve; the correlation coefficient of the calibration curve is greater than 0.9900, and the B / A of the AEB value is greater than 2.5.

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