Goat anti human fab

Serum concentrations of MxR and 3 × 1 were measured by ELISA. Briefly, Nunc maxsorp 96-well plates (Thermo Fisher, cat#442404) were coated with 100 ng of goat anti-human Fab (Jackson ImmunoResearch, cat#109-005-097) for 90 min at 4 °C. Wells were washed three times with 1×DPBS and then incubated with 1×DPBS containing 1% bovine serum albumin (Sigma-Aldrich, cat#A2153) for 1 h at room temperature. Antigen-coated plates were incubated with serum for 90 min at 4 °C. A standard curve was generated with serial two-fold dilutions of palivizumab. Wells were washed three times with 1×DPBS followed by a one-hour incubation with horseradish peroxidase-conjugated goat anti-human total Ig at a dilution of 1:6000 (Invitrogen, cat#31412). Wells were then washed four times with 1×DPBS followed by a 5–15 min incubation with TMB substrate (SeraCare, cat#5120-0053). Absorbance was measured at 405 nm using a Softmax Pro plate reader (Molecular Devices). The concentration of antibody in each sample was determined by reference to the standard curve and dilution factor.

Reactivity of recovered mAbs were tested against biotinylated tau peptides as previously described [37 (link)]. Briefly, tau peptides were captured on streptavidin-coated plates (Thermo Fisher Scientific) at 1 μg/ml in TBS and incubated for 2 h. Goat anti-human Fab (2 μg/ml, Jackson ImmunoResearch) to measure total IgG was used, and bovine actin (1 μg/ml TBS, Sigma) and irrelevant peptide were used to confirm specificity of the purified mAbs. ELISA plates were blocked and purified IgGs were diluted to 5 μg/ml in TBS/0.25% BSA and titrated (5-fold serial dilutions) against peptides. Plates were subsequently washed and goat anti-human IgG F(ab’)2 (Jackson ImmunoResearch) was used at 1:2000 dilution as secondary. Following incubation, plates were washed four times in TBS-T and developed with Sure Blue Reserve TMB Microwell Peroxidase Substrate (KPL) for approximately 2 min. The reaction was stopped by the addition of TMB Stop Solution and the absorbance at 450 nm was measured using an ELISA plate reader.