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. Here, we develop integrated fiber microneedle devices, an oral dosage form with tunable geometries and material configurations capable of both burst and sustained release to controlled depths in the oral mucosa. Integrated fiber microneedles administered to either the buccal or sublingual mucosa result in seroconversion and antigen-specific interferon-γ secretion in splenocytes. The dynamics and magnitude of the resulting immune response can be modulated by tuning microneedle release kinetics. Optimal microneedle geometry is site-specific, with longer microneedles eliciting greater immunogenicity in the buccal mucosa, and shorter microneedles eliciting greater immunogenicity in the sublingual mucosa. The Th1/Th2 phenotype of the resulting immune response is also dependent on integrated fiber microneedle length. Together, these results establish integrated fiber microneedles as a multifunctional delivery system for the oral mucosa and motivate further exploration using tunable delivery systems to better understand oral mucosal immunity.

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. Here, we have addressed the inaccuracy in AEB due to deviations from Poisson distribution in a digital ELISA for Aß-40 by changing the AEB calculation from a fixed threshold between digital counting and average normalized intensity to a smooth, continuous combination of digital counting and intensity. We addressed issues with determining the average product fluorescence intensity from single enzymes on beads by allowing outlier, high intensity arrays to be removed from average intensities, and by permitting the use of a wider range of arrays. These approaches improved the accuracy of a digital ELISA for tau protein that was affected by aggregated detection antibodies. We increased the dynamic range of a digital ELISA for IL-17A from AEB ∼25 to ∼130 by combining long and short exposure images at the product emission wavelength to create virtual images. The methods reported will significantly improve the accuracy and robustness of DBA based on imaging─such as single molecule arrays (Simoa)─and flow detection.

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%. Bead read efficiency was increased by: a) improving the loading of beads into arrays of microwells by combining capillary and magnetic forces in a method called magnetic-meniscus sweeping (MMS); b) using a centrifugal washer to minimize bead loss during the assay; and, c) improved optics and image analysis to enable the analysis of more microwells. Using this approach, we developed an assay for IL-17A with a limit of detection (LOD) of 0.7 aM, 437-fold more sensitive than standard digital ELISA. A digital ELISA with improved sensitivity was used to measure IL-17A in 100 serum and plasma samples with 100% detectability, compared to 51% for standard digital ELISA. Low numbers of capture beads yielded improved LODs for IL-12p70 (0.092 aM), p24 (9.1 aM), and interferon alpha (45.9 aM). IL-4 and PSA showed no improvements in sensitivity using fewer beads, primarily due to low antibody loading on beads and increased non-specific binding, respectively. The results were consistent with a kinetic model of binding that showed that combining capture antibodies with high on-rates with high antibodies per bead yields the greatest improvement in sensitivity.

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. By coupling each peptide to a unique assay capture antibody, this array of anchor antibodies enabled a customizable contact printed multiplex immunoassay workflow. As a proof of concept, we developed a 5-plex assay measuring interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 22 (IL-22), and tumor necrosis factor alpha (TNF-α). Measurements of these five analytes in serum and plasma correlated well between the method utilizing the anchor antibodies and peptides and the traditional capture antibody printing approach, with r2 values of 0.99, 0.93, 0.99, 0.96, and 0.75 for IL-5, IL-6, IL-10, IL-22, and TNFα, respectively. This approach makes customizable multiplex ultrasensitive ELISA available to laboratories without access to the precision printing instrumentation and will be useful for antibody screening, custom assay development, biomarker detection, and protein profiling for diagnostic applications.

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. CL from the arrays across the plate was collected in single images; long exposure times were used to maximize sensitivity, and short exposure times were used to extend the dynamic range at higher signals. Image analysis was used to determine the intensity of light from each spot in the array, and intensity was converted to concentration of protein via comparison to a calibration curve. To determine the intrinsic sensitivity of the CL ELISA array, streptavidin horseradish peroxidase (SA-HRP) was captured on an array spotted with biotinylated detection antibodies. The limit of detection (LOD) of SA-HRP was 105 aM, or 3200 enzymes per 50 µL. A single-plex assay for prostate specific antigen (PSA) was developed that had an LOD of 79 aM when the microtiter plate was shaken orbitally, comparable to the most sensitive immunoassays reported to date. Normalization of CL signals in the PSA assay to signal per molecule of SA-HRP showed that the efficiency of the shaken assay was ~40%. When the plates were not shaken, the efficiency was ~4.5%, i.e., ~9-fold lower than when shaken. To better understand the theoretical basis of the sensitivity of these assays, we developed COMSOL numerical models of the binding kinetics at the array for plates that were shaken orbitally and those not shaken. Experimental data from the orbitally shaken PSA assay were best modeled by inertial mixing in a three-layer system that included a 8-µm-thick concentration boundary layer. Experimental data from the unshaken PSA assay were well modeled by diffusion-limited kinetics. A single-plex assay for IL-10 was developed with an LOD of 69 aM or 1.5 fg/mL, and used to measure this cytokine in plasma and serum of 10 healthy individuals. A 5-plex assay for IL-5, IL-6, IL-10, IL-22, and TNF-α was developed with LODs of 56 aM, 237 aM, 69 aM, 88 aM, and 373 aM, respectively. The assay was used to measure these 5 cytokines in the plasma and serum of the same individuals. The correlation in concentration of IL-10 measured in single-plex and multiplex assays was good (r2 = 0.89; bias = 14.5%). The factors that result in the high sensitivity of CL ELISA arrays-mostly high signal to noise ratio of extended chemiluminescent imaging-are discussed. This multiplex CL ELISA could be used for sensitive profiling of multiple proteins for in vitro diagnostics and biomarker detection in the development of therapeutics.