Oral mucosal vaccination using integrated fiber microneedles.

The oral mucosa is an attractive site for immunization due to its accessibility and ability to elicit local and systemic immune responses. However, evaluating oral mucosal immunogenicity has proven challenging due to the physical barriers and immunological complexity of the oral mucosa. Microneedles can overcome these physical barriers, but previous work has been limited in the scope of microneedle delivery site, geometry, and release kinetics, all of which are expected to affect physiological responses.

Improving the Accuracy, Robustness, and Dynamic Range of Digital Bead Assays.

We report methods that improve the quantification of digital bead assays (DBA)─such as the digital enzyme-linked immunosorbent assay (ELISA)─that have found widespread use for high sensitivity measurement of proteins in clinical research and diagnostics. In digital ELISA, proteins are captured on beads, labeled with enzymes, individual beads are interrogated for activity from one or more enzymes, and the average number of enzymes per bead (AEB) is determined based on Poisson statistics. The widespread use of digital ELISA has revealed limitations to the original approaches to quantification that can lead to inaccurate AEB.

Digital enzyme-linked immunosorbent assays with sub-attomolar detection limits based on low numbers of capture beads combined with high efficiency bead analysis.

We report the development of digital enzyme-linked immunosorbent assays (ELISAs) based on single molecule arrays (Simoa) with improved sensitivities over conventional digital ELISA, enabling detection of proteins at sub-attomolar concentrations. The improvements in sensitivity were based on using fewer beads to capture the target proteins (≤5000 vs.∼500 000 beads) that increased the ratio of molecules to beads, and increasing the fraction of beads that were analyzed (bead read efficiency) from ∼5% to ∼50%.

Customizable Multiplex Antibody Array Immunoassays with Attomolar Sensitivities.

We have developed a customizable contact printed multiplex immunoassay capable of simultaneously measuring up to five analytes with attomolar sensitivities. This enzyme-linked immunosorbent assay (ELISA) was based on spotting different antibodies in a circular pattern at the bottom of a microtiter plate well. Unlike traditional antibody printing for ELISA that prints a capture antibody specific to a target of interest, in this ELISA we printed unique "anchor" antibodies at the well surface, each having a high affinity for a specific peptide target.

Sensitivity and binding kinetics of an ultra-sensitive chemiluminescent enzyme-linked immunosorbent assay at arrays of antibodies.

We have characterized the sensitivity and kinetics of a multiplex immunoassay system based on detection of chemiluminescence (CL) at arrays of antibodies. This enzyme-linked immunosorbent assay (ELISA) was based on the spotting of different antibodies in a circular pattern at the bottom of a well of a microtiter plate. Sandwich immunocomplexes within each spot were labeled with horse radish peroxidase, and CL was generated locally to each spot in the array from turnover of luminol substrate.