Recommendations on qPCR/ddPCR assay validation by GCC.

Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the development and validation of qPCR and ddPCR assays. Currently, however, there is limited regulatory guidance for the use of qPCR and a complete lack of any regulatory guidelines for the use of the newer ddPCR to support regulated bioanalysis. Hence, the Global CRO Council in Bioanalysis (GCC) has issued this White Paper to provide; 1) a consensus on the different validation parameters required to support qPCR/ddPCR assays; 2) a harmonized approach to their validation and 3) a consistent development of standard operating procedures (SOPs) for all the bioanalytical laboratories using these techniques.

Recommendations on ELISpot assay validation by the GCC.

Gene therapy, cell therapy and vaccine research have led to an increased need to perform cellular immunity testing in a regulated environment to ensure the safety and efficacy of these treatments. The most common method for the measurement of cellular immunity has been Enzyme-Linked Immunospot assays. However, there is a lack of regulatory guidance available discussing the recommendations for developing and validating these types of assays. Hence, the Global CRO Council has issued this white paper to provide a consensus on the different validation parameters required to support Enzyme-Linked Immunospot assays and a harmonized and consistent approach to Enzyme-Linked Immunospot validation among contract research organizations.

Serum/Plasma depletion with chicken immunoglobulin Y antibodies for proteomic analysis from multiple Mammalian species.

Plasma from different species is the most accessible and valuable source for biomarker discovery in clinical and animal samples. However, due to the high abundance of some proteins such as albumin and immunoglobulins, low-abundant proteins are often undetectable in proteomic analysis of plasma. We have established a plasma depletion scheme using chicken antibodies against various abundant proteins. This immunoaffinity purification procedure is able to deplete albumin across multiple species. The high binding capacity and specificity of the chicken antibody enables the efficient capture of its ligand from microliter volumes of plasma sample. The resulting two-dimensional gel analyses of the depleted and captured samples show significant enhancement of the low-abundant proteins and specific capture of the abundant ligand. By utilizing this sample preparation scheme, it is now possible to analyze the plasma proteome from multiple species in a potentially rapid and large-scale capacity for biomarker discovery, drug target discovery, and toxicology studies.

Depletion and fractionation technologies in plasma proteomic analysis.

This review intends to survey the traditional and current technologies in the depletion and subfractionation of plasma proteins for further analyses. The value of depletion aims to enrich low-abundant proteins by removing highly abundant proteins, such as albumin or immunoglobulin G, from plasma. With this approach, one can examine both the resulting high- and low-abundant protein fractions. The depleted protein population can be further subfractionated based on their isoelectric point ranges, creating a more discrete pool of proteins for detailed post-translational modification studies by methods such as 2D gel electrophoresis and mass spectrometry. The concept of divide to conquer will greatly enhance our ability to identify and characterize low-abundant proteins and cleaved peptides from plasma as important diagnostic markers or potential drug targets. This can potentially reverse the decline in the development of new plasma diagnostic tests.

Synthesis and pharmacological characterization of a potent, orally active p38 kinase inhibitor.

Inhibitors of the MAP kinase p38 provide a novel approach for the treatment of osteoporosis, inflammatory disorders, and cancer. We have identified N-(3-tert-butyl-1-methyl-5-pyrazolyl)-N'-(4-(4-pyridinylmethyl)phenyl)urea as a potent and selective p38 kinase inhibitor in biochemical and cellular assays. This compound is orally active in two acute models of cytokine release (TNF-induced IL-6 and LPS-induced TNF) and a chronic model of arthritis (20-day murine collagen-induced arthritis).