Encoded particle microfluidic platform for rapid multiplexed screening and characterization of aptamers against influenza A nucleoprotein.

Aptamers represent interesting bioreceptor alternatives to antibodies when developing a bioassay and are selected by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. After selection, an extensive characterization process is essential to verify the binding affinity and specificity of aptamer candidates, which is the most time-consuming and costly step. In this study, we assessed a new microfluidic platform, namely Evalution™, as a rapid and high throughput aptamer characterization platform. To do this, we first selected aptamers against influenza A nucleoprotein (infA NP) by performing magnetic bead-based SELEX. The selected aptamer candidates were subsequently screened using Evalution™ for their binding kinetics and specificity towards infA NP. All aptamers showed dissociation constants (KD) in the low nanomolar range (from 13 to 41 nM), and differential binding behavior towards control proteins, such as BSA and influenza B nucleoprotein (infB NP). Among 5 selected candidates, one aptamer (NP5) exhibited a significant discrimination between infA NP and infB NP and was further used to benchmark the kinetic analysis of Evalution™ (KD = 41 nM) with an SPR platform (KD = 17 nM). These results suggested that NP5 has the potential to be used for developing sensitive and infA NP specific aptamer-based assay. Moreover, the presented platform proved to be an efficient aptamer characterization tool for performing typical aptamer characterization experiments like binding kinetics (due to the real-time monitoring feature) and specificity assessment in a high-throughput manner due to the multiplexing capacity.

Fast multiplex analysis of antibodies in complex sample matrix using the microfluidic Evalution™ platform.

Biosensor development requires comprehensive research for establishing the optimal bioassay conditions that determine the final kinetics, sensitivity and specificity. Different systems have been developed to study bioreceptor-target interactions but they often have drawbacks, such as long hands-on time, low throughput, high sample consumption and high cost. In this work, the potential of the novel microfluidic Evalution™ platform has been evaluated for developing sandwich-based assays in a fast and high-throughput fashion. An immunoassay for the detection of influenza A nucleoprotein was used as a model system. Exploiting the platform's unique features, various typical bioassay parameters (e.g. aspecific binding between assay components, different reagent concentrations and incubation times) were tested for three capture antibodies in a simple and fast manner (2 runs of 80 min). The selected conditions, giving the highest signal-to-noise ratio, were directly employed on the same platform to detect nucleoprotein in buffer and nasopharyngeal swabs. Two antibodies with a higher dissociation constant (Ab11 and Ab12) required longer incubation times (60 min) for sensitive detection (limit of detection (LOD) of 0.48 and 0.26 ng mL-1, respectively) compared to an antibody with lower dissociation constant (LOD of 0.04 ng mL-1 for Ab66 within 30 min). Moreover, one antibody (Ab12) showed limited capacity to capture nucleoprotein directly in sample matrix. The obtained results were in accordance with previous studies performed on an ELISA and SPR platform with the same antibodies. This positions the Evalution™ platform as a reliable platform for fast and multiplex analysis of antibodies' performance both in buffer and complex sample matrices.

Bioassay Development for Ultrasensitive Detection of Influenza A Nucleoprotein Using Digital ELISA.

Flu is caused by the influenza virus that, due to mutations, keeps our body vulnerable for infections, making early diagnosis essential. Although immuno-based diagnostic tests are available, they have low sensitivity and reproducibility. In this paper, the prospect of detecting influenza A virus using digital ELISA has been studied. To appropriately select bioreceptors for this bioassay, seven commercial antibodies against influenza A nucleoprotein were methodically tested for their reactivity and binding affinity. The study has been performed on two markedly different platforms, being an enzyme-linked immunosorbent assay and a surface plasmon resonance system. The selected antibodies displayed completely different behavior on the two platforms and in various assay configurations. Surprisingly, the antibodies that showed overall good reactivity on both platforms had the highest dissociation constant among the tested antibodies, suggesting that, although important, binding affinity is not the only parameter to be considered when selecting antibodies. Moreover, only one antibody had the capacity to capture the nucleoprotein directly in lysis buffer used for releasing this viral protein, which might pose a huge advantage when developing assays with a fast time-to-result. This antibody was implemented on an in-house developed digital ELISA platform for ultrasensitive detection of recombinant nucleoprotein, reaching a detection limit of 4 ± 1 fM in buffer and 10 ± 2 fM in 10-fold diluted nasopharyngeal swabs, which is comparable to currently available fast molecular detection techniques. These results point to a great potential for ultrasensitive immuno-based influenza detection.

Separation of human immunoglobulin G subclasses on a protein A monolith column.

Monolithic columns have attracted significant attention for the purification of large biomolecules. In the present study, a step gradient elution method was evaluated for the separation of human immunoglobulin G (hIgG) into its subclasses on CIM (convective interaction media) r-protein A (recombinant protein A) monolithic column. hIgG was loaded onto the column and bound protein was eluted with a pH gradient. The subclass content of the eluted fractions was analyzed by enzyme-linked immunosorbent assay (ELISA). Results showed that separation of IgG3 from the other three subclasses can be successfully achieved with high selectivity (100%) and throughput on monolithic media. It was also revealed that enriched fractions of IgG1 and IgG2 could be obtained from purified hIgG in a 28min long chromatographic run. Three fractions with high IgG1 content (89.1%, 94.3% and 88.8%) were recovered. Furthermore, IgG2 was enriched to 64% successfully. A rapid step gradient elution scheme without any additives in buffers was proven to obtain enriched preparations of the two important subclasses with high throughput. The separation time can be reduced even more by increasing the flow rate without any loss in selectivity, which will be beneficial in industrial scale applications.