A key need for modern biology research is to make many quantitative molecular measurements on very small samples.  Our lab has been a leader in the development of multiplex assay technology that employs optically encoded microspheres and flow cytometry to make quantitative and sensitive molecular measurements. We have developed these approaches in the course of studies on the mechanisms of DNA repair, interactions between bacterial toxins and their receptors, susceptibility to virus induced cancer, and the detection of microbial pathogens.

Generic Approach to Molecular Analysis

Using Microspheres in Solid Supports

One current project, funded via an NIH Bioengineering Research Partnership, aims to develop a new platform for high throughput screening and highly multiplexed analysis based on Raman Flow Cytometry. This effort is targeted at developing diagnostics and therapeutics for microbial pathogens. As part of this project, we recently developed a rapid multiplexed approach to the screening of phage-display libraries. Other efforts are focused on quantitative molecular analysis of the immune and coagulation systems, as described below.

Raman spectra measured in flow.

The production of an effective antibody response depends on the production of enough antibodies with sufficient affinity to neutralize a pathogen. Effectiveness is often determined empirically in the form of protection, but it is desirable to understand protection in quantities terms of antigen-specific affinity. Popular titer-based methods of assessing antigen specific immune responses do not discern between antibody concentration and affinity, a critical distinction. We have developed an assay and analysis formalism that is able to measure both concentration and affinity across multiple isotypes simultaneously. Combined with our high throughput analysis capabilities, this approach has the potential to revolutionize our understanding of the development of the immune response, leading to a more complete understanding of the evolution of the antibody response and improved approaches for vaccine development. 

Analysis of antigen-specific antibodies: 

Titer vs affinity

The interaction between inflammation and coagulation in many diseases is well established, but molecular mechanisms and practical treatment strategies have been slow to emerge. Multiple lines of evidence implicate cell-derived membrane vesicles as key mediators of this interaction, but these very small (100 nm) circulating molecular assemblies are exceedingly difficult to study. We are developing instrumentation, including a  Microparticle Flow Cytometer, and associated methods to enable the quantitative enumeration and analysis of cell-derived microparticles, with the aim of developing mechanism-based biomarkers of disease. These efforts aim to open a new window on plasma components that mediated interaction between the immune and hemostasis systems.