第三代免疫分析技术 2019-02-03 11:58:57
图一. Evaluation of BCL-2:BIM complexes in HMCLs pre- and post-treatment with venetoclax.
BCL-2:BIM Complex Evaluation: BCL-2:BIM complexes were measured using an immunoassay platform. Briefly, 15 million MM cells were exposed to DMSO vehicle or 1 μM venetoclax for 24 hours and then processed for evaluation of the disruption of BCL-2:BIM complexes. Streptavidin coated plates was blocked for 16-24 hours with 40-150 μL blocking buffer (Mesoscale Discovery) while protein concentrations of cell lysates were adjusted to 4 mg/ml using cell lysis buffer. Anti-BCL-2 monoclonal antibody (Invitrogen) was labeled with 6:1 molar challenge ratio with ruthenium. Anti-BIM monoclonal antibody (Epitomics) was labeled with biotin at a 20:1 molar challenge ratio. Ruthenium-tagged anti-BCL-2 antibody was diluted to 1 μg/ml in incubation buffer and added to streptavidin-coated plates. An equal volume of cell lysates or BCL-2:BIM standard protein complexes were added to the plates and incubated at room temperature. Plates were washed with 0.5% polysorbate 20 in PBS followed by incubation with 1 μg/ml biotinylated anti-BIM antibody for 90 minutes at room temperature. Electrochemiluminescent signal intensity was measured on the Meso Scale Discovery SECTOR Imager 6000. Concentration of BCL-2:BIM complexes in samples treated with DMSO and venetoclax was determined by four-parameter fit logistic regression analysis based on the respective standard curves.
图二. MCL-1 inhibitor A-1210477 disrupts of MCL-1–BIM complexes.
After compound treatments, cells were lysed in buffer containing 1% CHAPS, 10 mM HEPES, and 150 mM NaCl with protease inhibitor cocktail (Roche, Indianapolis, IN, USA). In all, 100 μg of cell lysate was added to each well of a Meso Scale Discovery (Rockville, MD, USA) 96-well plate precoated with biotinylated anti-MCL-1 antibody (clone RC13) from Thermo Fisher Scientific. MCL-1-BIM complexes were detected with rabbit anti-BIM antibody (clone Y36) from Abcam, followed by Sulfo-tagged anti-rabbit antibody (Meso Scale Discovery; cat no. R32AB-5) and Meso Scale Discovery reading buffer with surfactant (cat. no. R92TC-2) according to the manufacturer’s recommendations. Each step was followed by washing three times with PBS-T (PBS+0.1% Tween-20).
案例三： MCL-1抑制剂S63845使用MSD检测cleaved PARP
图三. S63845 induces apoptosis of sensitive tumour derived cell lines.
案例四：MDM2抑制剂SAR405838 使用MSD检测MDM2 , p21 , p53, Cleaved Caspase 3, cleaved PARP
SAR405838: A novel and potent inhibitor of the MDM2:p53 axis for the treatment of dedifferentiated liposarcoma.
图四. Antitumor effects of SAR405838 in DDLPS xenograft models
For Mesoscale Discovery assays, protein analysis was performed after appropriate dilutions with following kits: MDM2 (K152FID), p21 (N45ZA-1), p53, Cleaved Caspase 3 and cleaved PARP (K15102D-1) with a detection on Sector Imager 2400. Results were normalized with total protein concentration.