Wetland cohesion is associated with increased probability of infection by the amphibian chytrid fungus Batrachochytrium dendrobatidis.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) poses a substantial threat to amphibian populations. Understanding the landscape conditions that facilitate Bd transmission and persistence is crucial for predicting Bd trends in amphibian populations. Here, we investigated the interactions between land use, wetland connectivity, and Bd occurrence and infection intensity. In northeastern Massachusetts, we sampled Pseudacris crucifer, Lithobates sylvaticus, L. clamitans, and L. pipiens from 24 sites. We found an overall 30.6% Bd prevalence at our sites, with prevalence differing among species. Bd occurrence increased with wetland-patch cohesion, potentially due to microclimate shifts from decreased forest or changes in host movement. Bd infection intensity was not mediated by landscape context. Overall, our results highlight the importance of landscape structure for Bd dynamics, suggesting that certain landscapes may facilitate transmission and harbor Bd more than others. To mitigate the impacts of Bd on amphibian populations, conservation efforts should account for interactions between Bd and landscape variables.

Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling.

Sortase A, a calcium-dependent transpeptidase derived from Staphylococcus aureus, is used in a broad range of applications, such as the conjugation of fluorescent dyes and other moieties to proteins or to the surface of eukaryotic cells. In vivo and cell-based applications of sortase have been somewhat limited by the large range of calcium concentrations, as well as by the often transient nature of protein-protein interactions in living systems. In order to use sortase A for cell labeling applications, we generated a new sortase A variant by combining multiple mutations to yield an enzyme that was both calcium-independent and highly active. This variant has enhanced activity for both N- and C-terminal labeling, as well as for cell surface modification under physiological conditions.

Screening individual hybridomas by microengraving to discover monoclonal antibodies.

The demand for monoclonal antibodies (mAbs) in biomedical research is significant, but the current methodologies used to discover them are both lengthy and costly. Consequently, the diversity of antibodies available for any particular antigen remains limited. Microengraving is a soft lithographic technique that provides a rapid and efficient alternative for discovering new mAbs. This protocol describes how to use microengraving to screen mouse hybridomas to establish new cell lines producing unique mAbs. Single cells from a polyclonal population are isolated into an array of microscale wells (approximately 10(5) cells per screen). The array is then used to print a protein microarray, where each element contains the antibodies captured from individual wells. The antibodies on the microarray are screened with antigens of interest, and mapped to the corresponding cells, which are then recovered from their microwells by micromanipulation. Screening and retrieval require approximately 1-3 d (9-12 d including the steps for preparing arrays of microwells).

Optimization of the surfaces used to capture antibodies from single hybridomas reduces the time required for microengraving.

The most common method for the generation of monoclonal antibodies involves the identification and isolation of hybridomas from polyclonal populations. The discovery of new antibodies for biochemical and immunohistochemical assays in a rapid and efficient manner, however, remains a challenge. Here, a series of experiments are described that realize significant improvements to an approach for screening large numbers of single cells to identify antigen-specific monoclonal antibodies in a high-throughput manner (10(5)-10(6) cells in less than 12 h). The soft lithographic process called microengraving yields microarrays of monoclonal antibodies that can be correlated to individual hybridomas; the cells can then be retrieved and expanded to establish new cell lines. The factors examined here included the glass slide used for the microarray, the buffer used to deposit capture antibodies onto the glass, the type of polyclonal antibodies used to capture the secreted antibodies, and the time required for microengraving. Compared to earlier reports of this method, these studies resulted in increased signal-to-noise ratios for individual elements in the microarrays produced, and a considerable decrease in the time required to produce one microarray from a set of cells (from 2-4 h to 3-10 min). These technical advances will improve the throughput and reduce the costs for this alternative to traditional screening by limiting serial dilution.

Profiling antibody responses by multiparametric analysis of primary B cells.

Determining the efficacy of a vaccine generally relies on measuring neutralizing antibodies in sera. This measure cannot elucidate the mechanisms responsible for the development of immunological memory at the cellular level, however. Quantitative profiles that detail the cellular origin, extent, and diversity of the humoral (antibody-based) immune response would improve both the assessment and development of vaccines. Here, we describe a novel approach to collect multiparametric datasets that describe the specificity, isotype, and apparent affinity of the antibodies secreted from large numbers of individual primary B cells (approximately 10(3)-10(4)). The antibody/antigen binding curves obtained by this approach can be used to classify closely related populations of cells using algorithms for data clustering, and the relationships among populations can be visualized graphically using affinity heatmaps. The technique described was used to evaluate the diversity of antigen-specific antibody-secreting cells generated during an in vivo humoral response to a series of immunizations designed to mimic a multipart vaccination. Profiles correlating primary antibody-producing cells with the molecular characteristics of their secreted antibodies should facilitate both the evaluation of candidate vaccines and, broadly, studies on the repertoires of antibodies generated in response to infectious or autoimmune diseases.