Method for detection or treatment of graft versus host disease
a graft-versus-host disease and reagent technology, applied in the field of graft-versus-host disease detection or treatment, can solve the problems of unavoidable genetic background and environment artifacts, new therapeutic methods are needed, and the graft-versus-host disease (gvhd) remains a major complication, and achieves accurate diagnosis.
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example 1
Bone Marrow Transplantation (BMT)
[0063]Acute GVHD was induced in mice via allogeneic bone marrow transplantation. BALB / c (H-2d) mice were used as the recipients, C57BL / 6 (H-2b) mice were used as the allogeneic BMT donors, and BALB / c (H-2d) mice were used as the syngeneic BMT donors. All mice were 7 to 12 weeks old (Sankyo Labo Service corporation, Japan).
[0064]On the day of BMT, the donor mice were sacrificed by cervical dislocation. Donor bone marrow cells were collected by flushing the shaft of the femur and tibia. The cells were placed in modified Eagle's medium containing 2% fetal calf serum / 1% penicillin-streptomycin, and prepared as a single cell suspension. The cells were rinsed with RPMI 1640 medium and resuspended in that same medium. The bone marrow cell inoculum was prepared to contain 2×107 bone marrow cells / 200 μL for allogeneic BMT and 1×106 bone marrow cells / 100 μL for syngeneic BMT.
[0065]The recipient BALB / c mice were raised on acidic water for at least 7 days before...
example 2
SELDI Protein Chip Array Analysis
[0073]To 10 μL of each plasma sample was added 20 μL of a solution comprising 9 mol / L urea and 10 g / L CHAPS in Tris-HCl (pH 7.4). The mixture was mixed for 15 min at 4° C. with a vortex mixer, and was then diluted to 1:40 in Tris-HCl. Eight-spot immobilized metal affinity capture arrays (IMAC-30) were activated with 50 mmol / L CuSO4. Diluted samples (50 μL) were applied to each spot of the protein chip array, and incubated for 1 h on a shaker. After washed with the same Tris-HCl, the chip was gently rinsed with water, and 0.5 μL of saturated sinapinic acid (SPA) was applied twice to each spot, and then air-dried. The mass / charge (m / z) spectra of the proteins bound to the chelated metal were measured using a Ciphergen Protein Biology System II Time-Of-Flight mass spectrometer (PBS II, Ciphergen Biosystems, Inc.) The data were calculated by averaging 65 laser shots obtained at a laser intensity of 200 and a detector sensitivity of 8.
[0074]Statistical An...
example 3
Separation of Proteins
[0084]The three most abundant proteins in the plasma (albumin, IgG, transferrin) were removed from the pooled plasma sample by immunodepletion chromatography (Multiple Affinity Removal Column MS-3, 4.6 mm ID×50 mm; Agilent). A 5-fold dilution of 50 μL of plasma in Buffer A (Agilent) was prepared and injected into the immunodepletion column. The flow-through fractions were collected and further separated by high-performance liquid chromatography (HPLC). The separation column used in HPLC was an Inertsil™ Ph column (5 μm, 4.6 mm ID×150 mm; GL Sciences, Japan). The elution gradient profile was as follows: (1) Elution solvent A: 2% ACN / 0.1% TFA, solvent B: 80% ACN / 0.1% TFA; (2) Linear gradient: 0 to 100% for solvent B for 50 min; flow rate 1.0 mL / min.
[0085]The fractions were collected at 30 sec intervals, 2 μL of each fraction was applied to an Au chip (Cipheragen), processed with an SPA matrix, and the composition of each fraction was monitored by SELDI-TOF MS. Th...
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