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

Abstract

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.

How Nanoparticles Transform Single Molecule Measurements into Quantitative Sensors

“…Instead, it is the off rate of detection antibodies targeting the analyte bound on a bead that determines the sensing performance and detection limits for the SiMoA. This has also been supported by other studies investigating the dependence of analytical performance of digital single molecule sensors upon the binding kinetics (on‐ and off‐rates) of capture and detection antibodies . When one factors the confinement in the well in the SiMoA experiment and the pore in the nanopore blockade experiment, where the analyte molecule may not be able to diffuse away at all, then the apparent unlimited lifetime of single molecule binding pairs becomes understandable.…”

Surface Plasmon Resonance: A Boon for Viral Diagnostics

“…Only one antibody was found to capture the nucleoprotein directly in the lysis buffer. Results provide another alternative for the ultrasensitive immunoassay-based detection of influenza virus (Leirs et al, 2016).…”

Towards the electrochemical diagnostic of influenza virus: development of a graphene–Au hybrid nanocomposite modified influenza virus biosensor based on neuraminidase activity

“…Although rapid detection of this virus is mandatory, present influenza diagnosis techniques cannot compete with the mutagenic behavior of the influenza virus. For example, viral culture, which was accepted as a golden standard method, needs two or three days to retrieve the results which is a kind of long time for the diagnosis of influenza virus [18][19][20] . On the other hand, quantitative polymerase chain reaction (qPCR), which is chosen frequently by the health personnel can be described as sensitive but relatively slow (took about 2 h).…”

“…On the other hand, quantitative polymerase chain reaction (qPCR), which is chosen frequently by the health personnel can be described as sensitive but relatively slow (took about 2 h). qPCR is also expensive and can be performed only with the specialists [20][21][22] . Apart from these methods, various immune based rapid tests have been fabricated recently to compensate the fast diagnosis need of the virus.…”

“…From this point of view, these tests manage to show the results in 15 to min. However, on the other hand, they lack off the sensitivity and the precision 20,21,23,24 . Electrochemical techniques are accepted as practical techniques for many applications 25,26 .…”