Kit for separating ADAMTS-13 protein in blood by using nickel magnetic beads

Abstract

The invention provides a kit for separating ADAMTS-13 protein in blood by using nickel magnetic beads. The kit is used for separating ADAMTS-13 protein in plasma or serum, and the method comprises the following steps: activating nickel magnetic beads in the kit, separating the magnetic beads by adhering to the wall, and sucking supernate; the nickel magnetic beads are specifically bound with ADAMTS-13 protein in plasma or serum in a binding buffer solution, the magnetic beads are subjected to adherent separation, and supernate is sucked; adding an elution buffer solution to clean the nickel magnetic beads, and removing non-specific binding proteins and impurities; 1% trifluoroacetic acid is added, the captured ADAMTS-13 protein on the nickel magnetic beads is dissolved, the magnetic beads are subjected to adherent separation, and supernate is sucked to obtain the separated ADAMTS-13 protein; the supernate can be directly used for MALDI-TOF-MS (Matrix-Assisted Laser Desorption / Ionization Time-Of-Flight Mass Spectrometry) mass spectrometry The kit for separating ADAMTS-13 protein in blood by nickel magnetic beads has the advantages of high sensitivity, strong specificity, wide application range, high flux, automation, low cost, rapidness, simplicity, convenience, flexibility in operation and small sample dosage, and can be used as a relatively important reference index clinically.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a kit for separating ADAMTS-13 protein from blood using nickel magnetic beads. More specifically, it relates to the use of a kit composed of nickel magnetic beads and related buffers for separating ADAMTS-13 protein from plasma or serum. Background Art

[0002] Nickel magnetic beads are high-capacity nickel-IMAC microbeads used for affinity purification of His-tagged fusion proteins. The surface of the magnetic beads is derivatized using nitrilotriacetic acid (NTA) chelating groups and carries divalent nickel ions (Ni 2+ 2+). Immobilized metal affinity chromatography (IMAC) microbeads offer high binding capacity and extremely low background. Nickel magnetic beads can be used in conjunction with a magnetic stand for manual operation or on an automated platform. Characteristics of nickel magnetic beads: 1. High capacity (particles less than 0.5 microns, binding capacity); 2. Low non-specific binding (pre-blocking the surface of the microbeads, the protocol provides optimized buffers for purification); 3. Fast (the protocol can be completed within 1 hour); 4. Scalable (can handle sample volumes from microliters to milliliters); 5. Versatile (can purify proteins under native or denaturing conditions); 6. Reagent compatible (can be used in conjunction with common cell lysis reagents and various buffer additives); 7. Multiple specifications (coupling of microbeads to proteins and downstream applications can be carried out manually or on an automated platform).

[0003] In 2001, Zheng XL et al. first cloned the ADAMTS-13 protein and classified it as a new member of the ADAMTS family, called the ADAMTS-13 protein. The full-length cDNA sequence of the ADAMTS-13 protein is 4.6 kb, and the full-length mRNA of the ADAMTS-13 protein in the liver was detected by RNA blotting. The ADAMTS-13 protein contains 1427 amino acids, including a hydrophobic signal peptide, a propeptide, a metalloprotease region, a disintegrin region, eight thrombospondin repeat motifs (TSP), a cysteine-rich region, a spacer region, and two CUB regions.

[0004] The nickel magnetic beads specifically bind to the ADAMTS-13 protein in plasma or serum. After a series of steps such as binding, enrichment, and elution, an ADAMTS-13 protein solution is obtained, which can be directly used for MALDI-TOF-MS mass spectrometry analysis or stored at -80 °C. Summary of the Invention

[0005] Based on the continuous development of the current MALDI-TOF-MS mass spectrometry technology in clinical applications, the inventors selected a series of protein purification conditions and finally determined the most suitable specific purification conditions for ADAMTS-13 protein at present, thereby providing a kit for separating ADAMTS-13 protein from blood using nickel magnetic beads. Among them, the nickel magnetic bead kit utilizes the principle of divalent nickel ions to adsorb ADAMTS-13 protein, and cooperates with a set of buffers suitable for MALDI-TOF-MS mass spectrometry analysis to enrich ADAMTS-13 protein from plasma or serum as much as possible, and accurately calculates the content of ADAMTS-13 protein in blood by establishing a standard curve with different concentrations of quality control substances.

[0006] Therefore, the objective of the present invention is to provide a kit for separating ADAMTS-13 protein from blood using nickel magnetic beads, which includes an activation buffer, an equilibration buffer, a binding buffer, an elution buffer, a substrate, a matrix, etc. suitable for nickel magnetic beads.

[0007] The second objective of the present invention is to provide the use of the ADAMTS-13 protein obtained by the above-mentioned kit for separating ADAMTS-13 protein from blood using nickel magnetic beads in MALDI-TOF-MS mass spectrometry analysis. Description of the Drawings

[0008] Figure 1 : A process diagram of nickel magnetic beads adsorbing ADAMTS-13 protein from blood and using magnetic separation to remove non-specific binding proteins and impurities.

[0009] Figure 2 : A mass spectrometry diagram of the ADAMTS-13 protein obtained by the kit for separating ADAMTS-13 protein from blood using nickel magnetic beads in the present invention in MALDI-TOF-MS mass spectrometry analysis. Detailed Embodiments

[0010] The following further details the present invention with specific examples.

[0011] Example 1: Reagent Composition of the Kit for Separating ADAMTS-13 Protein from Blood Using Nickel Magnetic Beads in the Present Invention

[0012] (1) Nickel magnetic beads

[0013] (2) Reaction buffer (5 mM Tris-HCl, 5 mM NaCl, pH 7.5, containing 1 mM BaCl2)

[0014] (3) Activation buffer (50 mM nickel sulfate)

[0015] (4) Elution buffer (1X PBS, 0.8 M sodium chloride, with 0.1% Triton X-100)

[0016] (5) Equilibration buffer (1 mM HEPES, pH 7.0)

[0017] (6) Binding buffer (1X PBS)

[0018] (7) Matrix (SPA: 5 mg / 100 μl ACN, 100 μl 1% TFA)

[0019] (8) Substrate

[0020] (9) Internal standard polypeptide

[0021] (10) PNP normal mixed plasma concentration curve (S1 - S7)

[0022] Example 2: Operating procedure of the kit for separating ADAMTS-13 protein from blood using nickel magnetic beads in the present invention

[0023] (1) PNP normal mixed plasma concentration curve (S1 - S7)

[0024] A 100% B 50% C 20% D 10% E 5% F 2.5% G 0%

[0025] Take 100 μl of PNP for standby, and take 50 μl of it as A; mix the other 50 μl with serum dilution buffer (100 mM NaCl, 0.1% BSA),

[0026] which is B; take 40 μl of B + 60 μl of Buffer = C; take 50 μl of C + 50 μl of Buffer = D; take 50 μl of D + 50 μl of Buffer = E; take 50 μl of E + 50 μl of Buffer = F; G is Buffer.

[0027] (2) First add 60 μl of reaction buffer (2) to an empty centrifuge tube, and then add 10 μl of quality control product and the serum of the patient to be tested respectively, and mix well.

[0028] (3) First add 25 μl of substrate (8) to a new empty centrifuge tube, and then take 10 μl of the diluted sample above and mix it with the substrate.

[0029] (4) Mark the centrifuge tube and place it on a small centrifuge tube rack and put it into a 37°C water bath for incubation for 60 minutes.

[0030] (5) After the incubation time is up, put the centrifuge tube into a 95°C thermostat for incubation for 2 minutes to terminate the reaction.

[0031] (6) Manually invert and mix the magnetic bead suspension thoroughly for 1 minute, and add 10 μl of nickel-tagged magnetic beads to an empty PCR tube.

[0032] (7) Add 100 μl of binding buffer (6) to each PCR tube. After incubating with shaking at room temperature for 5 minutes, place the PCR tubes on a magnetic rack to allow the magnetic beads to adhere to the wall for 1 minute. Discard the supernatant, and avoid touching the magnetic beads with the pipette tip to prevent sucking away the magnetic beads. Repeat once.

[0033] (8) Take out the centrifuge tubes in which the reaction has been terminated from the thermostat, and add 35 μl of the internal standard polypeptide (9) diluted 20-fold with binding buffer (6) to each tube, and mix well.

[0034] (9) Take 60 μl of the sample from the above centrifuge tubes and add it to the PCR tubes, and mix well with the magnetic beads. After incubating with shaking at room temperature for 30 minutes, place the PCR tubes on a magnetic rack to allow the magnetic beads to adhere to the wall, and discard the supernatant.

[0035] (10) Add 100 μl of elution buffer (4) to each PCR tube, incubate and shake at room temperature for 5 minutes, place the PCR tubes on a magnetic rack to allow the magnetic beads to adhere to the wall, discard the supernatant, and repeat once.

[0036] (11) Add 100 μl of equilibration buffer (5) to each PCR tube, place the PCR tubes on a magnetic rack to allow the magnetic beads to adhere to the wall, and discard the supernatant.

[0037] (12) Add 10 μl of 1% TFA (10 μl TFA + 990 μl water) to each PCR tube, pipette up and down 10 times to mix well, allow the magnetic beads to adhere to the wall for 2 minutes, and transfer the supernatant to a clean centrifuge tube.

[0038] (13) Add 10 μl of SPA matrix saturation solution to the PCR tubes containing the sample supernatant described above, mix well, then take 1 μl of the mixed sample and load it onto a mass spectrometry target plate, and dry it at room temperature.

[0039] Example 3: The ADAMTS-13 protein solution obtained using the kit for separating ADAMTS-13 protein from blood with nickel magnetic beads of the present invention was analyzed by MALDI-TOF-MS mass spectrometry.

[0040] (1) Load the mass spectrometry target plate with the ADAMTS-13 protein solution onto the MALDI-TOF-MS mass spectrometer for analysis and detection.

[0041] (2) The molecular weight of ADAMTS-13 protein presented on MALDI-TOF-MS mass spectrometry is about 7739.0 Da. Set the mass spectrometry parameters such as the molecular weight detection range of 20000 Da, the optimization range of 6000 - 10000 Da, a laser energy of 200 uJ, a sensitivity of 8, 120 collection times, an ion source voltage of 30 KV, and a detector voltage of 2950 V.

[0042] (3) Use the center point tool to select the correct peak for the internal control, correctly select the area under the curve of each peak and adjust the baseline, and select "Export" and "Peak Information" under the file. When prompted, select the following items: spectrum label, intensity, substance mass, and M / Z region. And save it to the known folder location.

[0043] (4) Open the file of each peak in Excel. Insert the peak information according to these instructions, calculate the standard curve and the results of the samples and controls. Check each column of the template to ensure that the correct formula is used. Before reporting the results, manually view each spectrum and the final results to check the calculation results.

[0044] (5) The standard curve of this detection was evaluated using PNP standards.

[0045] (6) Calculate the standard curve using the peak area ratio of the cleaved peptide to the internal standard (subtract the 0% PNP point from each standard or patient sample). Plot the ratio of each standard to the known dilution of PNP or the percentage of WHO, and then generate the standard curve using the linear regression equation: Y = Ax + b, where A = slope, b = y-axis intercept, y = diluted normal plasma (%), x = area ratio. The correlation coefficient (R) must be greater than or equal to 0.990 for the standard curve to be acceptable. If a point on the standard curve is unacceptable, it can be deleted and R recalculated. 2 If the correlation coefficient (R) of the entire curve is less than 0.990, the high-range and low-range standard curves will be plotted separately. The correlation coefficient (R) of the high and low curves must reach or exceed 0.950. If the standard curve does not meet these criteria, the run must be repeated.

[0046] (7) The control samples must be between 40 - 60% to be acceptable. If both samples are within the range, the average value should be used. If one of them is not within the range, the single data within this range can be used. If both controls are not within this range, they must be discarded.

[0047] (8) The acceptable performance range of the QC samples is shown in the following table.

[0048]

Claims

1. A kit for separating ADAMTS-13 protein from blood using nickel magnetic beads, comprising an activation buffer, a equilibration buffer, a binding buffer, an elution buffer, a substrate, a matrix, etc. suitable for nickel magnetic beads. The specific reagent composition is as follows: (1) Nickel magnetic beads (2) Reaction buffer (5 mM Tris-HCl, 5 mM NaCl, pH 7.5, containing 1 mM BaCl2) (3) Activation buffer (50 mM nickel sulfate) (4) Elution buffer (1X PBS, 0.8 M sodium chloride, with 0.1% Triton X-100) (5) Equilibration buffer (1 mM HEPES, pH 7.0) (6) Binding buffer (1X PBS) (7) Matrix (SPA: 5 mg / 100 μl ACN, 100 μl 1% TFA) (8) Substrate (9) Internal standard polypeptide.

2. Use of the ADAMTS-13 protein obtained by the kit of claim 1 for analysis on a MALDI-TOF-MS mass spectrometer.

Images