Evaluation of dried blood spots as alternative sampling material for serological detection of anti-SARS-CoV-2 antibodies using established ELISAs.

OBJECTIVES: During the current pandemic, antibody testing based on venous serum helps to determine whether the tested person has been previously infected with SARS-CoV-2. Alternatively, capillary blood can be taken via a finger prick (dried blood spots, DBS). In this study, paired DBS and venipuncture samples were tested using two serological assays to evaluate the usability of DBS for the detection of anti-SARS-CoV-2 antibodies. METHODS: Paired samples of DBS and venous serum were collected from 389 volunteers, of whom 75 had a recent PCR-confirmed SARS-CoV-2 infection, and tested for anti-SARS-CoV-2 IgG antibodies against both viral S1 and nucleocapsid protein (NCP) antigens using two ELISAs. Degree of agreement and correlation coefficients between ELISA results based on the two sampling methods were calculated. RESULTS: Results of DBS showed almost perfect agreement and high correlations with results from corresponding serum samples in both the S1-based ELISA and the NCP-based ELISA. CONCLUSIONS: ELISA results derived from DBS showed very high agreement to those obtained with serum, supposing adequate usability and robustness of DBS as sample material for detection of anti-SARS-CoV-2 antibodies. In the near future, large-scale epidemiological screening for antibodies against SARS-CoV-2 will be carried out. Since DBS reduce the strain on healthcare institutions regarding sample collection, they have a potential to facilitate efficient community- and population-based screening in the current SARS-CoV-2 pandemic.

The influence on the immunomodulatory effects of dying and dead cells of Annexin V.

Apoptotic and necrotic cells expose phosphatidylserine (PS). This membrane modification ensures a swift recognition and uptake by phagocytes of the dying and dead cells. Annexin V (AxV) preferentially binds to anionic phospholipids and thereby, modulates the clearance process. First, we analyzed the influence of AxV on the immunogenicity of apoptotic cells. The addition to apoptotic cells of AxV prior to their injection into mice increased their immunogenicity significantly. Next, we studied the influence of endogenous AxV on the allogeneic reaction against apoptotic and necrotic cells. To preserve heat-labile, short-lived "danger signals," we induced necrosis by mechanical stress. Wild-type mice showed a strong, allogeneic delayed-type hypersensitivity (DTH) reaction. In contrast, AxV-deficient animals showed almost no allogeneic DTH reaction, indicating that endogenous AxV increases the immune response against dead cells. Furthermore, AxV-deficient macrophages had a higher immunosuppressive potential in vitro. Next, we analyzed the influence of AxV on chronic macrophage infection with HIV-1, known to expose PS on its surface. The infectivity in human macrophages of HIV-1 was reduced significantly in the presence of AxV. Finally, we show that AxV also blocked the in vitro uptake by macrophages of primary necrotic cells. Similar to apoptotic cells, necrotic cells generated by heat treatment displayed an anti-inflammatory activity. In contrast, mechanical stress-induced necrotic cells led to a decreased secretion of IL-10, indicating a more inflammatory potential. From the experiments presented above, we conclude that AxV influences the clearance of several PS-exposing particles such as viruses, dying, and dead cells.

The low-throughput protein A adsorber: an immune modulatory device. Hypothesis for the mechanism of action in the treatment of rheumatoid arthritis.

To achieve specific removal of pathogenic antibodies (Ab) or immune complexes (IC), several adsorbers have been developed. We discuss the mode of action of low-throughput staphylococcal protein A (SPA) immunoadsorption. The SPA-based Prosorba apheresis is likely to modify some of the autoantibodies (autoAb) or IC. The low-throughput adsorber showed very limited adsorption capacity of circulating autoAb and/or circulating IC. Besides changes of humoral diagnostic parameters, cellular changes could be observed in the Prosorba-treated patients. These changes were rather similar to those that have been observed in a patient successfully treated with Ab against tumor necrosis factor alpha. We propose an adsorber-catalyzed conversion of small, tissue-penetrating, scarcely detectable, non-complement-binding, proinflammatory IgG-rheumatoid factor (RF)-based IC into the more readily phagocytosed species of IC: intermediate-sized, partially cryoprecipitable, non-tissue penetrating IC that are opsonized with complement. These IC are rather short-lived and could quickly be cleared by the body's scavenging system.

Early detection of apoptosis by staining of acid-treated apoptotic cells with FITC-labeled lectin from Narcissus pseudonarcissus.

BACKGROUND: Exposure of anionic phospholipids and modified carbohydrates are main parts of the apoptotic death program. Cells undergoing apoptosis can be identified by various methods, detecting surface changes or modifications of their organelles, respectively. We describe a method for the detection of early apoptosis by staining of cells with fluorescein isothiocyanate (FITC)-labeled lectin from Narcissus pseudonarcissus (NPn). METHODS: Apoptosis in cells or in cell lines was induced by various stimuli. To detect apoptosis the cells were stained with FITC-labeled lectin of NPn. After a short-term acid treatment they were analyzed by flow cytometry. RESULTS: The instability of the cytoplasmic membrane against acid and the binding of NPn were very early features of apoptotic cell death. The NPn lectin staining procedure detected apoptosis with high sensitivity. The staining was stable for at least 12 h. CONCLUSIONS: The method described in this study is suitable for the detection of the very early phases of apoptosis. The NPn lectin staining after short-term acid treatment can, therefore, be added to the list of reliable tools for the research of cell death.