Objective The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic challenges national health systems and the global economy. Monitoring of infection rates and seroprevalence can guide public health measures to combat the pandemic. This depends on reliable tests on active and former infections. Here, we set out to develop and validate a specific and sensitive enzyme linked immunosorbent assay (ELISA) for detection of anti-SARS-CoV-2 antibody levels. Methods In our ELISA, we used SARS-CoV-2 receptor-binding domain (RBD) and a stabilized version of the spike (S) ectodomain as antigens. We assessed sera from patients infected with seasonal coronaviruses, SARS-CoV-2 and controls. We determined and monitored IgM-, IgA-and IgG-antibody responses towards these antigens. In addition, for a panel of 22 sera, virus neutralization and ELISA parameters were measured and correlated. Results The RBD-based ELISA detected SARS-CoV-2-directed antibodies, did not cross-react with seasonal coronavirus antibodies and correlated with virus neutralization (R 2 = 0.89). Seroconversion started at 5 days after symptom onset and led to robust antibody levels at 10 days after symptom onset. We demonstrate high specificity (99.3%; N = 1000) and sensitivity (92% for IgA, 96% for IgG and 98% for IgM; > 10 days after PCR-proven infection; N = 53) in serum. Conclusions With the described RBD-based ELISA protocol, we provide a reliable test for seroepidemiological surveys. Due to high specificity and strong correlation with virus neutralization, the RBD ELISA holds great potential to become a preferred tool to assess thresholds of protective immunity after infection and vaccination.

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Cultured cells of Eschscholzia californica respond to a yeast glycoprotein elicitor by producing benzophenanthridine alkaloids, which are excreted into the cell wall and the outer medium. These compounds, preferentially sanguinarine, are efficient phytoalexins because of their ability to intercalate double-stranded DNA (dsDNA), penetrate membranes and inhibit various enzymes containing SHgroups. Externally added sanguinarine is rapidly taken up by intact cells and converted to dihydrosanguinarine, which is substituted intracellularly according to the biosynthetic route. A 29.5 kDa soluble enzyme that catalyses the reduction of sanguinarine and chelerythrine by either NADPH or NADH has been isolated and purified to homogeneity. Benzophenanthridines that accumulate in the outer medium, mainly 10-OH-chelerythrine, chelirubine and macarpine, are converted by the isolated enzyme and by intact cells at much slower rates than sanguinarine. The cellular capacity of uptake and conversion of sanguinarine largely surpasses the rate of alkaloid production. We conclude that the sanguinarine produced by intact cells, after excretion and binding to cell wall elements, is rapidly reabsorbed and reduced to the less toxic dihydrosanguinarine, which then undergoes further biosynthetic reactions. This recycling process would allow the presence of the toxic phytoalexin at the cellular surface without taking the risk of injuring the producing cell.

Sotatercept (formerly ACE-011) is a glycosylated, dimeric fusion protein composed of the extracellular domain of the human activin receptor type IIA (ActRIIA) and the Fc region of human IgG1. The protein-based drug candidate acts as a ligand trap which competitively binds to activin A and other members of the transforming growth factor beta superfamily, thus blocking signalling through ActRIIA. Since the inhibition of activin A was found to significantly increase bone formation and quality, Sotatercept was originally developed for the treatment of diseases involving bone loss. But as the protein therapeutic also stimulates erythropoiesis by a mechanism independent of the EPO receptor, it has been evaluated for the treatment of anaemia in rare blood diseases such as beta thalassemia. Due to its positive effects on erythropoiesis and bone formation, Sotatercept may also be misused as performance-enhancing agent in sports. Within this study, two complementary detection assays for Sotatercept and related ActRIIA-Fc fusion proteins in serum samples were developed. While the first assay combines affinity purification and Western blotting to generically detect ActRIIA-Fc fusion proteins irrespective of their amino acid sequence, the liquid chromatography-high resolution mass spectrometry (LC-HRMS) method is highly specific for proteolytic peptides originating from the receptor and Fc domain of Sotatercept. Both approaches can readily be modified to include other pharmaceutical proteins such as therapeutic antibodies, and serve as proof-of-concept for the capability of the approach to detect TGF-β inhibitors and Fc fusion proteins in doping control serum samples. Copyright © 2016 John Wiley & Sons, Ltd.

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Sanguinarine reductase is a plant enzyme that prevents the cytotoxic effects of benzophenanthridine alkaloids, which are the main phytoalexins of Papaveraceae. The enzyme catalyzes the reduction of sanguinarine, the most toxic benzophenanthridine, which re-enters the cytoplasm after its primary accumulation in the cell wall region has reached a threshold concentration. We present the sequence of the gene and protein of sanguinarine reductase isolated from cell cultures of Eschscholzia californica. High sequence similarities indicate that the enzyme evolved from a plant-specific branch of the ubiquitous Rossmann fold NAD(P)H/NAD(P)؉ binding reductases, with NADP-dependent epimerases or hydroxysteroid reductases as the most likely ancestors. Based on the x-ray structure of a close homolog, a three-dimensional model of the spatial conformation and catalytic site of sanguinarine reductase was established and used for in silico screening of known three-dimensional structures. Surprisingly, the enzyme shares high structural similarity with enzymes of human and bacterial origin, which have similar functions as the plant homologs but bear little amino acid sequence similarity. Using site-directed mutagenesis, a series of recombinant enzymes was generated and assayed to reveal the impact of individual amino acids and peptides in the catalytic process. It appears that relatively few innovations were required to generate this selective catalyst for alkaloid detoxication, notably an insertion of 13 amino acids and the generation of a novel catalytic triad of Cys-Asp-His were sufficient.

Human erythropoietin (hEPO) is an erythropoiesis stimulating hormone frequently employed in antianemia therapy. Its capability to increase the amount of red blood cells however makes hEPO and its derivatives also attractive to dishonest athletes aiming at an artificial and illicit enhancement of their endurance performance. A major objective of the international antidoping fight is the elimination of drug misuse and prevention of severe adverse effects caused by nontherapeutic administrations of highly potent drugs. The emergence of novel and innovative erythropoietin-mimetic agents (EMAs) has been continuously growing in the last years, and the option of using dedicated monoclonal antibodies (mAb) for analytical and sample preparation approaches is gradually reaching limits. In the present study the common ability and property of all EMAs, to bind on the human erythropoietin receptor (hEPOR), is therefore exploited. An alternative methodology to isolate and analyze EMAs, in particular endogenous EPO and the recombinant forms EPOzeta, darbepoetin alfa, and C.E.R.A., from human urine is described, employing conventional ultrafiltration for preconcentration of the target analytes followed by EMA-specific isolation via hEPOR-bound magnetic beads. Analytical data were generated by means of gel-based electrophoretic analysis and nanoliquid chromatography/high resolution/high accuracy tandem mass spectrometry. Limits of detection enabled by the established sample preparation protocols were approximately 20 pg/mL for EPOzeta, 30 pg/mL for darbepoetin alfa, and 80 pg/mL for C.E.R.A.

AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside) arguably provides performance-enhancing properties even in the absence of physical exercise and, therefore, the substance is banned in elite sports since 2009. Due to the natural presence of AICAR in human blood and urine, uncovering the misuse by direct qualitative analysis is not possible. Entering the circulation, the riboside is immediately incorporated into red blood cells (RBCs) and transformed into the corresponding ribotide (5'-monophosphate) form. Within the present study, an analytical method was developed to determine AICAR-ribotide concentrations in RBC concentrates by means of liquid chromatography-tandem mass spectrometry. The method was validated enabling quantitative result interpretation considering the parameters specificity, precision (intra- and interday), linearity, recovery, accuracy (LOD/LOQ), stability and ion suppression. By analysing 99 RBC samples of young athletes, normal physiological levels of AICAR-ribotide were determined (10-500 ng/mL), and individual levels were found to be stable for several days. Employing in vitro incubation experiments with AICAR riboside in fresh whole blood samples, the ribotide concentrations were observed to increase significantly within 30 min from baseline to 1-10 μg/mL. These levels are considered conserved for the lifetime of the erythrocyte and, thus, the results of the in vitro model strongly support the hypothesis that measuring abnormally high AICAR-ribotide concentrations in RBC of elite athletes has the potential to uncover the misuse of this substance for a long period of time.