Subject(s)
Postpartum Hemorrhage , Humans , Pilot Projects , Female , Pregnancy , Postpartum Hemorrhage/diagnosis , Postpartum Hemorrhage/therapy , AdultABSTRACT
Monoclonal antibodies are a main player in modern drug discovery. Many antibody screening formats exist, each with specific advantages and limitations. Nonetheless, it remains challenging to screen antibodies for the binding of cell-surface receptors (the most important class of all drug targets) or for the binding to target cells rather than purified proteins. Here, we present a high-throughput droplet microfluidics approach employing dual-color normalized fluorescence readout to detect antibody binding. This enables us to obtain quantitative data on target cell recognition, using as little as 33 fg of IgG per assay. Starting with an excess of hybridoma cells releasing unspecific antibodies, individual clones secreting specific binders (of target cells co-encapsulated into droplets) could be enriched 220-fold after sorting 80,000 clones in a single experiment. This opens the way for therapeutic antibody discovery, especially since the single-cell approach is in principle also applicable to primary human plasma cells.
Subject(s)
Antibodies/analysis , Microfluidics/methods , Single-Cell Analysis/methods , Antibodies/metabolism , Cell Membrane/metabolism , Cell Survival , Fluorescence , Humans , Hybridomas/metabolism , Immunoassay , K562 Cells , Protein BindingABSTRACT
The use of microfluidic droplets has become a powerful tool for the screening and manipulation of cells. However, currently this is restricted to assays involving a single cell type. Studies on the interaction of different cells (e.g. in immunology) as well as the screening of antibody-secreting cells in assays requiring an additional reporter cell, have not yet been successfully demonstrated. Based on Poisson statistics, the probability for the generation of droplets hosting exactly one cell of two different types is just 13.5%. To overcome this limitation, we have developed an approach in which different cell types are stained with different fluorescent dyes. Subsequent to encapsulation into droplets, the resulting emulsion is injected into a very compact sorting device allowing for analysis at high magnification and fixation of the cells close to the focal plane. By applying dual-color sorting, this furthermore enables the specific collection and analysis of droplets with exactly two different cells. Our approach shows an efficiency of up to 86.7% (more than 97% when also considering droplets hosting one or more cells of each type), and, hence, should pave the way for a variety of cell-based assays in droplets.
Subject(s)
Cell Separation/instrumentation , Lab-On-A-Chip Devices , Color , Hybridomas/cytologyABSTRACT
Surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS) can provide positive identification of an analyte or an analyte mixture with high sensitivity and selectivity. Better understanding of the theory and advances in the understanding of the practice have led to the development of practical applications in which the unique advantages of SERS/SERRS have been used to provide effective solutions to difficult analytical problems. This review presents a basic theory and illustrates the way in which SERS/SERRS has been developed for practical use.
Subject(s)
Biotechnology , Nanotechnology , Spectrum Analysis, Raman , Surface Plasmon Resonance , Molecular Diagnostic Techniques , NanostructuresABSTRACT
Optimisation of colloidal properties allows Surface Enhanced Raman Scattering (SERS) to be recorded from a range of analytes at 1546 nm, demonstrating the potential of SERS for use in a wavelength region of particular value for applications such as homeland security.
