Technical library

タンパク質のリン酸化は、多くのシグナル伝達カスケードで不可欠な可逆的な反応です。シグナル伝達カスケードの特性評価は、リン酸化抗体を用いたイムノブロッティングでその特異性と親和性を十分に評価しなければなりません。ここではリン酸化抗体の特異性を評価するために、キャピラリー電気泳動ベースのイムノアッセイ（CEIA）で自動化と定量を活用して、1回のライセート調製でリン酸化抗体の特異性を評価する簡単なアッセイを説明します。薬剤での刺激前後のライセートサンプルを分子量ごとに分離し、UV活性化架橋反応によりキャピラリーの内壁に固定しました。固定したタンパク質をラムダフォスファターゼで1時間処理後、化学発光または蛍光イムノアッセイを行い、各活性化経路のリン酸化タンパク質に結合する抗体の特異性を調べました。ここで説明する方法を用いると、リン酸化タンパク質に対する抗体の特異性を評価するのに、複数のライセートや追加のブロットが不要になります。

During adeno-associated virus (AAV) manufacture, critical quality attributes must be monitored including the presence, identity, and purity of viral vector proteins. Traditionally, the identity and purity of these proteins is monitored by Western blot using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). However, SDS-PAGE is notoriously challenging and labor-intensive. Additionally, AAVs are limited in sample size as they are difficult to manufacture, and demand outstrips supply. Here, we have developed a method to monitor the purification of AAV2 using automated capillary electrophoresis followed by immunoassay and total protein detection directly in the capillary, eliminating the need for SDS-PAGE. AAV2 was purified from HEK293 cell lysate using affinity chromatography, and the steps of the purification process (load, flow-through, wash and elution) were monitored by the capillary-based immunoassay. VP1, VP2 and VP3 capsid proteins were resolved and identified either individually or simultaneously, depending on the AAV antibody used for detection, and the total protein assay monitored the presence of impurities. The sensitivity of this assay reduced sample size down to 3 µL of sample, corresponding to approximately 400 pg or 1x10⁸ genomic copies loaded per well. We anticipate that this in-capillary immunoassay and total protein detection can replace traditional SDS-PAGE methods in AAV manufacturing workflows.

The tumor microenvironment (TME) is a complex mixture of cancerous and non-cancerous cells, including immune cells like T-cells, macrophages, and neutrophils. The TME plays a key role in tumorigenesis and metastasis, and it has recently been recognized that it can dramatically shape a response to therapy. Thus, there is a pressing need to accurately identify and quantify the variety of cell types in any given TME. However, studying the TME presents major challenges. For example, the heterogeneity of the environment requires sensitive and high-resolution techniques to parse subpopulations of different cell types. This challenge is compounded by the severely limited sample size that can be obtained from donor tissues. To address these challenges, we use an in-capillary immunoassay with small sample sizes (3 µL) to identify immune cells commonly found in the TME. We also leverage single-cell Western to uncover trends in population heterogeneity. Human peripheral blood mononuclear cells (PBMCs) were differentiated into dendritic cells (DCs) and regulatory T cells (Tregs), and natural killer (NK) cells were expanded from isolated NK cells. These samples were then analyzed by in-capillary immunoassay and single-cell Western. These analyses revealed the identification and characterization of cell types, at both the single-cell and population level, based on the differential expression of protein biomarkers. Specifically, in-capillary immunoassay analysis identified mature populations by CD209 for DCs, a CD56+/CD3- phenotype for NK cells, and CD25 and Foxp3 expression for Tregs. Analysis of single cells provided further detail within these populations, for example, we observed FoxP3low and FoxP3high subpopulations in Tregs, and an unexpectedly large (81%) CD56-/CD3- subpopulation in undifferentiated PBMCs, suggesting the presence of other cell subtypes. We anticipate that the small sample size, automation, single-cell resolution, and multiplexing ability of these assays collectively will enable a more efficient and deeper characterization of the TME not possible with traditional immunoassays like Western blot and flow cytometry.

Alzheimer disease (AD) affects mainly people over the age of 65 years, suffering from different clinical symptoms such as progressive decline in memory, thinking, language, and learning capacity. The toxic role of beta amyloid peptide (Ab) has now shifted from insoluble Ab fibrils to smaller, soluble oligomeric Ab aggregates (AβO). Many evidences suggest that the neurodegenerative process would be due to the interaction of AβO with binding targets, activation of stress kinases, hyperphosphorylation of tau protein, caspase activation, loss of synapse, neuronal death, loss of cholinergic function, generation of reactive intermediates of oxygen (oxidative stress), or glutamate excitotoxicity. Urgent need for efficient new therapies is high, but could only be successful with an extensively comprehension of AβO degeneration process. In the present work, based on an in vitro primary cell culture treated with AβO preparation, we have carefully studied the cytopathological effects of AβO on neuronal death and then we have investigated the effect of 17-beta Estradiol (β-estr) on the degeneration process induced by AβO. Briefly we used rat cortical neurons (from E15). The cells were seeded in 96-well plates and intoxicated with AβO solution after 11 days of culture for 24 hours. β-estr was used at 100 nM (final concentration) and was added as pretreatment (1h before injuries). A co-incubation with selective inhibitors was performed for the mechanistic study. In parallel, western blotting (WB) analysis was done to quantify protein levels and their activation. We showed that β-estr was able to significantly protect neurons as well as glial cells from degeneration decreasing the caspase 3 activation and the massive mitochondrial stress (induced by AβO). Preservation of neurite network and synapsis integrity was also observed. Moreover, the large hyperphosphorylation of tau protein induced by AβO was significantly reduced with β-Estr. A mechanistic study was also performed co-incubating inhibitors of main survival pathways to try to better understand the mode of action of β-estr and the pathway involved in the AβO toxicity. We showed that the effects of -Estr were fully abolished blocking the MEK pathway as well as the DNA repair pathway (PARP-g) or the mitochondrial anti-apoptotic pathway (Bcl2). Interestingly the effect was inexisting coincubating β-estr with TrK receptor or Ras/Raf inhibitors showing the predominant role of growth factors paythway in its neuroprotective effect. Finally, we showed a large inactivation of AKT protein in presence of AβO that was reversed even over activated in presence of β-Estr.