A multiplex serological assay for the characterization of IgG immune response to SARS-CoV-2.

In the fight against SARS-COV-2, the development of serological assays based on different antigenic domains represent a versatile tool to get a comprehensive picture of the immune response or differentiate infection from vaccination beyond simple diagnosis. Here we use a combination of the Nucleoprotein (NP), the Spike 1 (S1) and Spike 2 (S2) subunits, and the receptor binding domain (RBD) and N-terminal domain (NTD) of the Spike antigens from the CoViDiag® multiplex IgG assay, to follow the immune response to SARS-CoV-2 infection over a long time period and depending on disease severity. Using a panel of 209 sera collected from 61 patients up to eight months after infection, we observed that most patients develop an immune response against multiple viral epitope, but anti-S2 antibodies seemed to last longer. For all the tested IgGs, we have found higher responses for hospitalized patients than for non-hospitalized ones. Moreover the combination of the five different IgG responses increased the correlation to the neutralizing antibody titers than if considered individually. Multiplex immunoassays have the potential to improve diagnostic performances, especially for ancient infection or mild form of the disease presenting weaker antibody responses. Also the combined detection of anti-NP and anti-Spike-derived domains can be useful to differentiate vaccination from viral infection and accurately assess the antibody potential to neutralize the virus.

micro-RNA 21 detection with a limit of 2 pM in 1 min using a size-accordable concentration module operated by electrohydrodynamic actuation.

We present a fluorimetry-based technology for micro-RNA-21 (miR-21) sensing based on the concentration of miR-molecular beacon (MB) complexes and flushing of unbound MB. This concentration module consists of a microfluidic channel with the shape of a funnel operated with electrohydrodynamic actuation. We report a limit of detection of 2 pM in less than 1 min for miR-21 alone, and then demonstrate that miR-21 levels, measured in fine needle biopsy samples, from patients with pancreatic cancer correlate with the reference technique of reverse-transcription polymerase chain reaction (RT-PCR). Altogether, this technology has promising clinical performances for the follow-up of patients with cancer.

Agrodiag PorCoV: A multiplex immunoassay for the differential diagnosis of porcine enteric coronaviruses.

Three different porcine enteric coronaviruses (PECs), i.e., porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine Deltacoronavirus (PDCoV) are currently circulating in U.S. commercial swine herds. Differential diagnosis of PECs relies on laboratory methods. This study describes the development of an ELISA-like multiplex planar immunoassay based on virus-specific recombinant S1 proteins printed in an array of spots at the bottom of a 96-well microplate for simultaneous detection differential serodiagnosis of PEDV, TGEV, PDCoV in a single sample. The technology overall format and working principle is similar to the solid-phase standard ELISA. After the three typical incubation steps, the reaction was visualized as blue spots which intensity correlated with antibody levels to specific viral antigen target in the array. The diagnostic performance of the assay was evaluated on known status serum samples (n = 480) collected over time (day post-inoculation -7, 0, 7, 14, 21, 28, 35, and 42) from pigs inoculated with PEDV, TGEV Purdue, TGEV Miller, PDCoV (USA/IL/2014), or mock inoculated with culture media under experimental conditions. Antigen-specific cut-offs were selected to ensure 100% diagnostic and analytical specificity for each given antigen target. The overall diagnostic sensitivity was 92% (44/48 positives, 95% confidence interval (CI) 98,100) for PEDV S1, 100% (95/95 positives, 95% CI 98, 100) for TGEV S1, and 98% (47/48 positives, 95% CI 97, 100) for PDCoV S1. The results of this study demonstrate that the AgroDiag PEC multiplex immunoassay is an efficient and reliable test for differential detection and serodiagnosis of PEDV, TGEV and PDCoV.

µLAS: Sizing of expanded trinucleotide repeats with femtomolar sensitivity in less than 5 minutes.

We present µLAS, a lab-on-chip system that concentrates, separates, and detects DNA fragments in a single module. µLAS speeds up DNA size analysis in minutes using femtomolar amounts of amplified DNA. Here we tested the relevance of µLAS for sizing expanded trinucleotide repeats, which cause over 20 different neurological and neuromuscular disorders. Because the length of trinucleotide repeats correlates with the severity of the diseases, it is crucial to be able to size repeat tract length accurately and efficiently. Expanded trinucleotide repeats are however genetically unstable and difficult to amplify. Thus, the amount of amplified material to work with is often limited, making its analysis labor-intensive. We report the detection of heterogeneous allele lengths in 8 samples from myotonic dystrophy type 1 and Huntington disease patients with up to 750 CAG/CTG repeats in five minutes or less. The high sensitivity of the method allowed us to minimize the number of amplification cycles and thus reduce amplification artefacts without compromising the detection of the expanded allele. These results suggest that µLAS can speed up routine molecular biology applications of repetitive sequences and may improve the molecular diagnostic of expanded repeat disorders.

DNA separation and enrichment using electro-hydrodynamic bidirectional flows in viscoelastic liquids.

DNA size separation followed by purification and enrichment constitute essential operations for genetic engineering. These processes are mostly carried out using DNA electrophoresis in gels or in polymer solutions, a well-established yet lengthy technique which has been notably improved using Lab-on-Chip technologies. So far, innovations for DNA separation or enrichment have been mostly undertaken separately, and we present an approach that allows us to perform these two processes simultaneously for DNA fragments spanning 0.2-50 kilo base pairs (kbp) in length. Our technology involves an electric field and a counter hydrodynamic flow in viscoelastic liquids, in which we show the occurrence of transverse forces oriented toward the walls. These forces increase with DNA molecular weight (MW) and hence induce a progressive reduction in DNA migration speed that triggers size separation in microfluidic channels as well as in capillaries. The separation of MW markers in the range 1-50 kbp is achieved in 15 minutes, thus outperforming gel electrophoresis that takes ∼3 hours for this sample. Furthermore, the use of a funnel, where electric and flow fields are modulated spatially, enables us to adjust the transverse forces so as to stall the motion of DNA molecules at a position where they accumulate at factors of up to 1000 per minute. In this configuration, we establish that the operations of DNA enrichment and separation can be carried out simultaneously for the bands of a DNA MW marker between 0.2-1.5 kbp diluted at 0.02 ng µL(-1) in 30 s. Altogether, our technology, which can readily be integrated as an in-line module in Lab-on-Chips, offers unique opportunities for sample preparation and analysis of minute genomic samples.