Alexa fluor 647 conjugated anti human igg h l

A flow cytometry live cell‐based assay was used to detect the binding of patient serum antibodies against surface human D2R, an accepted method of neuronal antibody detection,⁴ and was performed as previously described.¹⁷, ¹⁸, ¹⁹, ²¹ Briefly, transfected live HEK293 cells expressing D2R were incubated with serum (1:50),¹⁰³ followed by staining with Alexa Fluor 647‐conjugated anti‐human IgG (H + L; Thermo Fisher Scientific, Waltham, MA, USA). Cells were acquired live using the high‐throughput system on a five‐laser BD LSR II Flow Cytometer and analysed with FlowJo 10.4.1, Excel and GraphPad Prism. The threshold was calculated as the mean + 3SD of the control cohort, and a patient was reported positive for D2R antibodies if they were above this threshold in at least two of three independent experiments. Only 23 of 24 patients were tested as the serum of one patient was not available. Antibodies against other dopamine receptor subtypes were not detected in our previous study and therefore were not tested.¹⁷

The assay to detect patient serum antibodies against SARS-CoV-2 antigens using flow cytometry has been previously described in detail (35 (link)). Briefly, HEK293 cells were transfected to transiently express SARS-CoV-2 full-length Spike (Wuhan-1 D614), Membrane and Envelope proteins. Diluted patient serum was added to the cells, followed by adding AlexaFluor 647-conjugated anti-human IgG (H+L) (Thermo Fisher Scientific) or anti-human IgM (A21249, Thermo Fisher Scientific). The LSRII flow cytometer (BD Biosciences, USA) was used to acquire the cells and patients were confirmed SARS-CoV-2 antibody positive if, in at least two of three quality-controlled experiments, their median fluorescence intensity (ΔMFI   =   MFI transfected cells − MFI untransfected cells) was above the positive threshold (mean ΔMFI + 4SD of 24 pre-pandemic controls). Data were analysed using FlowJo 10.4.1 (BD Biosciences), Excel (Microsoft, USA), and GraphPad Prism (GraphPad Software, USA).