Especially in the cases of some of the sera derived from ARDS patients (sera A5 and A52), a high titer of spike-specific IgAs could be detected while the IgG titer was low. of ARDS sera, which are characterized by a significantly higher portion of RBD-binding IgA antibodies, there is a obvious correlation between the neutralization assays and the ELISA data. In contrast to this, a less obvious correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was Ketoconazole observed, which might be explained from the heterogeneity of the antibodies. To conclude, for less complex immune seraas in instances of non-ARDS seracombinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, dedication of the stability of the antigenCantibody complex, and competition of the RBD-ACE2 binding symbolize alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for any BSL3 facility. Keywords: COVID-19, SARS-CoV-2, convalescent sera, ARDS, neutralization assay, humoral immune response 1. Intro After its emergence in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread all over the world. SARS-CoV-2 is an enveloped disease. The positive-sense, single-stranded RNA genome has a size of approximately 30 kb. In the family using a 10 kDA Amicon Centrifugal Filter (Merck Millipore, Burlington, MA, USA) was performed. 2.3. Plaque Reduction Neutralization Test (PRNT50) For the PRNT (plaque reduction neutralization test) Vero Er cells were used; 6 105 of Vero E6 cells per well of a 6-well plate (Greiner Bio-One, Kremsmnster, Austria) were seeded and incubated for 24 h at 37 C. For the analysis, the sera were 2-collapse serially diluted (1:20 to 1 1:640) and incubated with 80 PFU of the early isolate (MRS) in a total volume of 100 L at 37 C for 1 h. In the next step, the medium was eliminated and replaced by 1 mL of new DMEM without FCS. The complete virusCserum blend was Ketoconazole added to the cells and incubated at 37 C for 1 h. During incubation, the 6-well plate was agitated every 15 min. After incubation, the supernatant was eliminated and 2 mL of 0.4% SeaPlaque Agarose (Biozym, Hessisch-Oldendorf, Germany) in DMEM containing 5% FCS, 1% penicillin/streptomycin and 1% L-glutamine was given to the infected cells. After 2 days, the cells were fixed for 20 min having a 4% formaldehyde remedy in PBS and stained with 1 mL 0.1% crystal violet in 20% ethanol for 15 min. Finally, after removal of the staining remedy, the wells were washed with 2 mL distilled water and the plaques were counted. The PRNT titer was determined FGF2 based on a 50% reduction in plaque counts (PRNT50). The PRNT50 titer was chosen relating to a WHO guideline [19]. As compared with higher cutoffs (PRNT90), the dedication of the PRNT50 enables more accurate results as the titers are based on the linear part of the titration curve. 2.4. Ketoconazole Pseudotype-Based Neutralization Assay For generation of lentiviral vectors HEK293T cells were co-transfected with HIV-1 gag/pol, rev, the luciferase-encoding lentiviral vector genome and the SARS-CoV-2 delta 19 spike (#”type”:”entrez-nucleotide”,”attrs”:”text”:”MN908947″,”term_id”:”1798172431″,”term_text”:”MN908947″MN908947), using Lipofectamine? 2000 (Thermo Fisher, Darmstadt, Germany), as described previously [20]. The SARS-CoV-2 gene was commercially synthesized (Eurofins, Ebersberg, Germany) and cloned into a pIRES-GFP vector, as explained before [21]. After harvest and concentration by ultracentrifugation, vector particles were stored at ?80 C. Pseudotyped vectors and serially diluted human being serum (1:60 to 1 1:14,580) were incubated in triplicates for 45 min at 37 C and then used to transduce HEK293T-hACE2 cells [22]. After 48 h, Britelite plus luciferase substrate (PerkinElmer, Waltham, MA, USA) was added to measure luciferase activity. For each sample, the reciprocal area under the curve (AUC) value was determined. The AUC ideals were calibrated to the WHO international standard and international devices/mL are indicated as neutralization activity. 2.5. Sera Convalescent sera (Table 1) from hospitalized individuals suffering from ARDS or not were provided by the RWTH Centralized Biomaterial Standard bank (RWTH cBMB) of the Medical Faculty of RWTH Aachen University or college. The two sera (N11801 and N11802) from.