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.

Concordance with established tests and reproducibility of results obtained with a new single antigen chip array for HLA antibody detection (HISTO SPOT® HLA AB).

The purpose of this study was to analyze performance of a new single antigen chip array system (HISTO SPOT® HLA AB) developed for HLA antibody detection and compare with results obtained using single antigen Luminex-based systems and serum samples from the Eurotransplant external proficiency testing scheme. Results were analysed from 11 independent Eurotransplant laboratories using HISTO SPOT® HLA AB utilising the Eurotransplant external proficiency testing (EPT) sera and these were compared to published results from 67 labs using the Luminex-based technologies. In addition, QC results from different batches of the test were analysed. Generally, concordance of results with the results from the Luminex technique was good. With the Luminex tests more consensus results and more questionable results were found than with the HISTO SPOT® HLA AB test. Within the HISTO SPOT® HLA AB testing group we found a discrepancy rate from the consensus of 2.9% for the EPT sera which is far below the 25% allowed to pass the quality test and only slightly higher than for the Luminex single antigen tests with 1.2%. The average global coefficient of variation (CV) of the mean signal (raw data) for the HISTO SPOT® HLA AB test was 13% which is lower than the values reported for Luminex tests in the literature. The average global CV for the signal/background ratio was higher with 28%. In the present study, the mean signal is the best parameter to compare results between labs and the new HISTO SPOT® HLA AB test is at least as good in terms of signal reproducibility as the Luminex tests. In conclusion, the HISTO SPOT® HLA AB test is a good alternative to be used in addition or instead of the Luminex tests in clinical labs.

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.

Humoral immunity links Candida albicans infection and celiac disease.

OBJECTIVE: The protein Hwp1, expressed on the pathogenic phase of Candida albicans, presents sequence analogy with the gluten protein gliadin and is also a substrate for transglutaminase. This had led to the suggestion that C. albicans infection (CI) may be a triggering factor for Celiac disease (CeD) onset. We investigated cross-immune reactivity between CeD and CI. METHODS: Serum IgG levels against recombinant Hwp1 and serological markers of CeD were measured in 87 CeD patients, 41 CI patients, and 98 healthy controls (HC). IgA and IgG were also measured in 20 individuals from each of these groups using microchips sensitized with 38 peptides designed from the N-terminal of Hwp1. RESULTS: CI and CeD patients had higher levels of anti-Hwp1 (p=0.0005 and p=0.004) and anti-gliadin (p=0.002 and p=0.0009) antibodies than HC but there was no significant difference between CeD and CI patients. CeD and CI patients had higher levels of anti-transglutaminase IgA than HC (p=0.0001 and p=0.0039). During CI, the increase in anti-Hwp1 paralleled the increase in anti-gliadin antibodies. Microchip analysis showed that CeD patients were more reactive against some Hwp1 peptides than CI patients, and that some deamidated peptides were more reactive than their native analogs. Binding of IgG from CeD patients to Hwp1 peptides was inhibited by γIII gliadin peptides. CONCLUSIONS: Humoral cross-reactivity between Hwp1 and gliadin was observed during CeD and CI. Increased reactivity to Hwp1 deamidated peptide suggests that transglutaminase is involved in this interplay. These results support the hypothesis that CI may trigger CeD onset in genetically-susceptible individuals.

Inhibition of protein-protein interactions in Plasmodium falciparum: future drug targets.

The rapid development by malaria parasites of resistance to almost all the chemotherapeutic agents so far used for their control means that constant efforts to develop new drugs are necessary. In this review, we propose that the exploration of protein-protein interactions as a new strategy to identify antimalarial drug targets is an attractive and a promising area of research. Nevertheless, one of the most important criteria is that the targeted gene should encode an essential protein within a complex that is able to affect parasite survival. Recently, our research on the biology of Plasmodium falciparum allowed us to identify the interaction of Protein Phosphatase type 1 and actin with two essential partners, PfLRR1 and PfLRR7 respectively, both of which belong to the Leucine Rich Repeat (LRR) protein family. LRR-containing proteins are composed of several consensus LRR motifs LXLXXNXL (where X is any amino acid) that provide sites for the assembly of protein interactions. The LRR combines structural versatility, adaptability and more importantly a high degree of interaction specificity. In addition, it has been shown that a single mutation in a particular LRR motif abolishes the protein-protein interaction and contributes to the expression of severe pathology in humans. This clearly infers that blocking the interaction related to 'hot spots' of LRR motifs can be considered as good targets to block parasite growth and development. Thus, the inhibition of protein-protein interactions by peptides, peptidomimetics or small-molecule inhibitors that interfere with binding domains can contribute to defining new potential drug targets.

Peptide microarrays on bisphenol A polycarbonate.

Microarrays are frequently prepared on microscope glass slides. However, glass substrates can break or cut and thus can lead to the contamination of the manipulator during the analysis of biological samples. Alternately, bisphenol A polycarbonate (PC) is shock-resistant and, in addition, is easily eliminated by incineration. We show here that PC is a useful substrate for peptide microarray preparation. We describe in particular the preparation of peptide microarrays on PC using semicarbazide-functionalized silica nanoparticles and in situ semicarbazone ligation with glyoxylyl-peptides. The microarrays were used for the detection of antibodies using fluorescence detection.

In situ ligation between peptides and silica nanoparticles for making peptide microarrays on polycarbonate.

Bisphenol A polycarbonate (PC) is emerging as an interesting alternative to silicon oxide substrates for making microarrays. We show that the printing of peptide/nanoparticle mixtures allows the creation of complex peptide microarrays. Semicarbazone ligation was used for linking the peptides to the nanoparticles. The reaction occurred probably after printing due to solvent evaporation and to the in situ concentration of the reagents. Peptide microarrays were used successfully for the specific capture of purified antibodies or of antibodies from serum.

Imaging of protein layers with an optical microscope for the characterization of peptide microarrays.

Solid-phase assays play a crucial role today in biological studies. These assays are based on the immobilization of probe molecules on a surface, which are able to capture specifically soluble receptors. In particular, peptide microarrays have emerged as powerful tools in a variety of applications. In this context, optical techniques that allow imaging of nanometer-thick biomolecular films, and thereby the characterization of microarrays, are of great interest. For this purpose, we used a recently disclosed wide-field optical imaging technique of surface nanostructures called Sarfus, which is based on the use of a standard optical microscope and antireflection substrates. We demonstrate here that this technique allows the imaging of the protein layers that result from the specific capture of antibodies by arrayed peptide probes with a spatial resolution of 0.45 microm. The relationship between the thickness of the antibody layer and peptide or antibody concentration was examined.

Chemical micropatterning of polycarbonate for site-specific peptide immobilization and biomolecular interactions.

Polycarbonate (PC) is a useful substrate for the preparation of microfluidic devices. Recently, its utility in bioanalysis has attracted much attention owing to the possibility of using compact discs as platforms for the high-throughput analysis of biomolecular interactions. In this article we report a novel method for the chemical micropatterning of polycarbonate based on the printing of functionalized silica nanoparticles. The semicarbazide groups present on the surface of the nanoparticles were used for the site-specific semicarbazone ligation of unprotected peptides derivatized by an alpha-oxoaldehyde group. The peptide micropatterns permitted the specific capture of antibodies. We report also the characterization of micropatterns on PC by using a wide-field optical imaging technique called Sarfus; this allows the detection of nm-thick films by using nonreflecting PC substrates and an optical microscope working with reflected differential interference contrast. The method described here is an easy way to modify polycarbonate surfaces for biomolecular interaction studies and should stimulate the use of PC for developing plastic biosensors.