Detection of residual T7 RNA polymerase used in mRNA in vitro transcription by Simple Western.

Therapeutic messenger RNA (mRNA) has been demonstrated as a scalable and versatile vaccine platform for the rapid development and manufacture of new vaccine candidates. mRNA is synthesized enzymatically through in vitro transcription (IVT) using bacteriophage T7 RNA polymerase (T7 RNAP), a 99 kDa protein with high binding affinity for the promoter sequence and a low error rate. Post-IVT, mRNA is purified to remove impurities, but if T7 RNAP is insufficiently cleared, undesirable clinical side effects may result. Therefore, it is important to quantitate T7 RNAP concentrations in IVT and process intermediates to understand clearance during downstream purification. A high-throughput T7 RNAP assay was developed using Simple Western (SW), a capillary immunoassay technology, to quantitate concentrations as low as 5.3 ng/mL with good precision and accuracy. Compared to existing T7 RNAP immunoassays or total protein assays such as bicinchoninic acid assays or Bradford, the SW T7 RNAP assay is specific to T7 RNAP, requires <10 µL of sample volume, and consists of minimal sample handling and hands-on time. This work highlights the development and optimization of a highly sensitive and robust T7 RNAP quantitation assay using the SW platform.

Mechanism underlying severe deficiency of plasma ADAMTS-13 activity in immune thrombotic thrombocytopenic purpura.

BACKGROUND: Immune-mediated thrombotic thrombocytopenic purpura is caused by autoantibodies against ADAMTS-13, a plasma enzyme that cleaves von Willebrand factor. However, the mechanism resulting in severe deficiency of plasma ADAMTS-13 activity remains controversial. OBJECTIVES: To determine the mechanism of autoantibody-mediated severe deficiency of plasma ADAMTS13 activity in immune-mediated thrombotic thrombocytopenic purpura. METHODS: Fluorescence resonance energy transfer-VWF73 was used to determine plasma ADAMTS-13 activity. Enzyme-linked immunosorbent assay (ELISA) was used to determine anti-ADAMTS-13 immunoglobulin G. ELISA and capillary electrophoresis-based Western blotting were employed to assess plasma ADAMTS-13 antigen. RESULTS: We showed that plasma ADAMTS-13 antigen levels varied substantially in the samples collected on admission despite all showing plasma ADAMTS-13 activity of <10 IU/dL (or <10% of normal level) using either ELISA or Western blotting. More severe deficiency of plasma ADAMTS-13 antigen (<10%) was detected in admission samples by ELISA than by capillary Western blotting. There was a significant but moderate correlation between plasma ADAMTS-13 activity and ADAMTS-13 antigen by either assay method, suggesting that severe deficiency of plasma ADAMTS-13 activity is not entirely associated with low levels of ADAMTS-13 antigen. CONCLUSION: We conclude that severe deficiency of plasma ADAMTS-13 activity primarily resulted from antibody-mediated inhibition, but the accelerated clearance of plasma ADAMTS-13 antigen via immune complexes may also contribute significantly to severe deficiency of plasma ADAMTS-13 activity in a subset of patients with acute immune-mediated thrombotic thrombocytopenic purpura.

Quantification of AMPA receptor subunits and RNA editing-related proteins in the J20 mouse model of Alzheimer's disease by capillary western blotting.

Introduction: Accurate modelling of molecular changes in Alzheimer's disease (AD) dementia is crucial for understanding the mechanisms driving neuronal pathology and for developing treatments. Synaptic dysfunction has long been implicated as a mechanism underpinning memory dysfunction in AD and may result in part from changes in adenosine deaminase acting on RNA (ADAR) mediated RNA editing of the GluA2 subunit of AMPA receptors and changes in AMPA receptor function at the post synaptic cleft. However, few studies have investigated changes in proteins which influence RNA editing and notably, AD studies that focus on studying changes in protein expression, rather than changes in mRNA, often use traditional western blotting. Methods: Here, we demonstrate the value of automated capillary western blotting to investigate the protein expression of AMPA receptor subunits (GluA1-4), the ADAR RNA editing proteins (ADAR1-3), and proteins known to regulate RNA editing (PIN1, WWP2, FXR1P, and CREB1), in the J20 AD mouse model. We describe extensive optimisation and validation of the automated capillary western blotting method, demonstrating the use of total protein to normalise protein load, in addition to characterising the optimal protein/antibody concentrations to ensure accurate protein quantification. Following this, we assessed changes in proteins of interest in the hippocampus of 44-week-old J20 AD mice. Results: We observed an increase in the expression of ADAR1 p110 and GluA3 and a decrease in ADAR2 in the hippocampus of 44-week-old J20 mice. These changes signify a shift in the balance of proteins that play a critical role at the synapse. Regression analysis revealed unique J20-specific correlations between changes in AMPA receptor subunits, ADAR enzymes, and proteins that regulate ADAR stability in J20 mice, highlighting potential mechanisms mediating RNA-editing changes found in AD. Discussion: Our findings in J20 mice generally reflect changes seen in the human AD brain. This study underlines the importance of novel techniques, like automated capillary western blotting, to assess protein expression in AD. It also provides further evidence to support the hypothesis that a dysregulation in RNA editing-related proteins may play a role in the initiation and/or progression of AD.