Polyclonal rabbit anti human igm

Western blotting was performed to determine the immunogenic region of Pap31 and SCS-α. Both the purified complete proteins and their respective 3 truncated constructs were electrophoresed on 15% SDS-PAGE gels and electrotransferred onto a PVDF membrane (GE Healthcare, Buckinghamshire, UK) using a Bio-Rad Trans-Blot SD Cell apparatus. A blocking step for 1 h at room temperature using 5% skim milk in PBS was performed. The membranes were immunoblotted during 90 min with sera (1:50 dilution in Tris-buffered saline [TBS]-Tween with 1% bovine serum albumin [BSA]). Western blottings with at least 10 sera from exposed people were performed for both Pap31 and SCS-α. A commercial negative control (DAKO X0939) consisting of a pool of at least 30 sera from healthy blood donors was used. After washing 3 times with TBS-0.05% Tween20) for10 min, the membranes were incubated for 1 h with a polyclonal rabbit anti-human IgM 1:1000 dilution in TBS-T with 1% BSA conjugated with peroxidase (Dako, Glostrup, Denmark) using o-phenylenediamine tablets as substrate (Sigma, St. Louis, MO, USA).

MaxiSorp microtiter plates (Nunc) were coated with 28 ng/well polyclonal rabbit anti-human IgM (Dako) in carbonate buffer pH 9.6 [35 (link)]. Diluted human serum samples were added for 45 min at 37°C. Bound IgM antibodies were then detected with the strep-tagged E proteins and Strep-Tactin-labeled HRP (IBA GmbH, Göttingen, Germany). For the quantitative Zika IgM ELISA, a Zika virus post-infection serum sample (TBE-pre-vaccinated patient) was included as a standard, set at 1,000 IgM arbitrary units (S3 Fig). Quantification of unknown samples was performed with dilutions within the linear range of the standard serum. The cut-off (75 IgM units) was determined with 32 flavivirus negative samples (S3 Fig). In the case of the RB IgM ELISA, the cut-off was calculated as the mean absorbance value of the negative samples plus 3 standard deviations.