Rapid mycobacteria drug susceptibility testing using Gel Microdrop (GMD) Growth Assay and flow cytometry.

Control of multi-drug-resistant tuberculosis has been hampered by the lack of simple, rapid and sensitive methods for assessing bacterial growth and antimicrobial susceptibility. Due to the increasing incidence and high frequency of mutations, it is unlikely that culture methods will disappear in the foreseeable future. Therefore, the need to modernize methods for rapid detection of viable clinical isolates, at a minimum as a gold standard, will persist.

Comparison of results using the gel microdrop cytokine secretion assay with ELISPOT and intracellular cytokine staining assay.

The gel microdrop (GMD) secretion assay involves encapsulating single cells in a biotinylated agarose matrix, addition of a streptavidin bridge, diffusion of a biotinylated capture antibody, and detection of secreted molecules using a fluorescently labeled reporter antibody. Using flow cytometry, encapsulated cells can be analyzed or recovered based on cell type and secretory profile. Using murine Th2 cell line D10.

Isolation of rare isotype switch variants in hybridoma cell lines using an agarose gel microdrop-based protein secretion assay.

Using gel microdrop (GMD) encapsulation technology and fluorescence-activated cell storing (FACS), we have developed a rapid, sensitive, and reliable method for discriminating and recovering rare isotypic switch variants in hybridoma cell lines. Using the GMD-based IgSwitch assay, a novel approach for isolating subpopulations of IgG-secreting hybridoma cells present at a frequency of approximately 1-10 in 10(6), we successfully isolated spontaneous and in vitro-induced isotypic switch variants in less than half the time required for conventional sublining. The effectiveness and specificity of the assay are demonstrated.

Antigen-driven peripheral immune tolerance: suppression of experimental autoimmmune encephalomyelitis and collagen-induced arthritis by aerosol administration of myelin basic protein or type II collagen.

Antigen-driven tolerance is an effective method of suppressing cell-mediated immune responses. We have previously demonstrated that exposure of gut-associated lymphoid tissue to myelin basic protein (MBP) via oral administration suppresses experimental autoimmune encephalomyelitis (EAE). To further study presentation of antigen to the immune system by mucosal surfaces as a method of antigen-driven tolerance, the effect of inhalation of MBP was investigated.