SARS-CoV-2 antibody progression and neutralizing potential in mild symptomatic COVID-19 patients - a comparative long term post-infection study.

Background: Since December 2019, SARS-CoV-2 has been keeping the world in suspense. Rapid tests, molecular diagnosis of acute infections, and vaccination campaigns with vaccines are building blocks of strategic pandemic control worldwide. For laboratory diagnostics, the quantification of the antibody titer of convalescents and vaccinated patients is thus increasingly coming to the fore. Methods: Here we present an evaluation on the comparability of five serological tests on a cohort of 13 patients with mild COVID-19 disease. Also participants who were vaccinated after recovery were included in this study. All common immune methods (ELISA, CLIA, PETIA) and SARS-CoV-2 specific antigens (N-, S1- and RBD-) were specifically tracked and directly compared for up to 455 days. The titer of recovered participants was also set to the degree of symptoms during infection and the occurrence of Long-COVID. In addition, relative comparability of different serological tests, all standardized to WHO, was set in reference to the neutralizing potential of the corresponding participants. Findings: The individual immune responses over 455 days after a mild SARS-CoV-2 infection remain stable, in contrast to vaccinated participants. All sero-tests reveal comparable performance and dynamics during the study and compared well to a surrogate neutralization test. Conclusion: The information presented here will help clinicians in the daily laboratory work in the selection and evaluation of different serological tests offered. The data also will support in respect of a sero-test-based neutralization cutoff.

Metal ions and phosphatidylinositol 4,5-bisphosphate as interacting effectors of α-type plant phospholipase D.

Plant phospholipases D (PLD) are typically characterized by a C2 domain with at least two Ca2+ binding sites. In vitro, the predominantly expressed α-type PLDs need 20-100 mM CaCl2 for optimum activity, whereas the essential activator of ß- or γ-type PLDs, phosphatidylinositol 4,5-bisphosphate (PIP2), plays a secondary role. In the present paper, we have studied the interplay between PIP2 and metal ion activation of the well-known α-type PLD from cabbage (PLDα). With mixed micelles containing phosphatidyl-p-nitrophenol as substrate, PIP2-concentrations in the nanomolar range are able to activate the enzyme in addition to the essential Ca2+ activation. Mg2+ ions are able to replace Ca2+ ions but they do not activate PLDα. Rather, they abolish the activation of the enzyme by Ca2+ ions in the absence, but not in the presence, of PIP2. The presence of PIP2 causes a shift in the pH optimum of PLDα activity to the acidic range. Employing fluorescence measurements and replacing Ca2+ by Tb3+ ions, confirmed the presence of two metal ion-binding sites, in which the one of lower affinity proved crucial for PLD activation. Moreover, we have generated a homology model of the C2 domain of this enzyme, which was used for Molecular Dynamics (MD) simulations and docking studies. As is common for C2 domains, it shows two antiparallel ß-sheets consisting of four ß-strands each and loop regions that harbor two Ca2+ binding sites. Based on the findings of the MD simulation, one of the bound Ca2+ ions is coordinated by five amino acid residues. The second Ca2+ ion induces a loop movement upon its binding to three amino acid residues. Docking studies with PIP2 reveal, in addition to the previously postulated PIP2-binding site in the middle of the ß-sheet structure, another PIP2-binding site near the two Ca2+ ions, which is in accordance with the experimental interplay of PIP2, Ca2+ and Mg2+ ions.

Lanthanides as substitutes for calcium ions in the activation of plant α-type phospholipase D.

Most types of phospholipase D (PLD) from plants contain a C2 domain and are activated by Ca(2+) ions. In this study, other metal ions such as Mg(2+), La(3+), Ce(3+), Tb(3+) and Y(3+) were examined as effectors of recombinantly produced α-type PLD from white cabbage. All the rare earth ions were able to substitute for Ca(2+). The activation curves and displacement experiments reflect a 10- to 50-fold higher affinity of PLD for these ions than for Ca(2+); however, the maximum activity attained only 36% of that in the presence of Ca(2+). Mg(2+) displaced Ca(2+) without being able to activate PLD. All ions were bound to the substrate micelles consisting of phosphatidyl-p-nitrophenol, Triton X-100 and SDS (1:8:1, by mole). The affinity of rare earth ions to the micelles was 100-fold higher than that of Ca(2+) and Mg(2+). A conformational change of the enzyme induced by the low affinity but specific binding of Ca(2+) ions is concluded to be essential for maximal PLD activity. As demonstrated by the measurement of Tb(3+) fluorescence, the substitution of Ca(2+) by rare earth ions provides a new avenue for studying the enigmatic role of Ca(2+) ions in the modulation of PLD activity in plants.

Fe(III)-resorcylate as a spectrophotometric probe for phospholipid-cation interactions.

A simple spectrophotometric microplate assay that allows quantification of the interaction between phospholipids and metal ions or other small cationic compounds has been developed. The assay is based on the competition of the phospholipids for the Fe(3+) ion in the purple-colored Fe(III)-γ-resorcylate complex and for other cations. To compare the binding affinities of several cation-phospholipid interactions, K0.5 values were derived from binding curves constructed by determination of the absorbance of the Fe(III)-γ-resorcylate at 490 nm as a function of the cation concentration. The assay was used to analyze the binding of lanthanide ions, calcium ions, and amines (hydrochlorides of ethanolamine, spermidine, and hexyltrimethylammonium chloride) to small unilamellar vesicles (SUVs) and mixed micelles containing anionic lipids such as phosphatidic acid and phosphatidyl-p-nitrophenol. The method was evaluated by fluorescence measurements with Eu(3+) ions as tracer. Lanthanide ions such as La(3+) and Ce(3+) ions showed K0.5 values smaller by one to two orders of magnitude compared with Ca(2+) ions. In the presence of increasing amounts of detergents such as Triton X-100, the method also reflected transitions from SUVs to micelles. The binding capacity for metal ions was higher for phospholipid-containing micelles than for the corresponding SUVs.