Goat anti human igg f ab 2

Protein extraction, denaturation, and Western blotting were performed as previously described (29 (link), 60 (link), 61 (link)). Membranes were blotted with anti–phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9101, Cell Signaling) and anti–p44/42 MAPK (Erk1/2) (4695, Cell Signaling). Images were captured with ImageQuantLAS 4000 (GE Healthcare Life Sciences). For detection of D2AP11-TCE expression, goat anti-human IgG F(ab′)₂ (109-005-006, Jackson ImmunoResearch Laboratories Inc.) and donkey anti-goat secondary antibodies (926-32214, LI-COR) were used, and membranes were scanned using a LI-COR Odyssey CLx imager.

Streptavidin-coated 96-well plates (Thermo Fisher) were washed four times with TBS-T, followed by incubation overnight at 4 °C with 400 nM of the panel of tau-specific biotinylated peptides in Table S3. Plates were washed four times the following day with TBS-T and subsequently blocked for 1 h with 2.5% BSA in TBS. Human anti-tau antibodies were added at 2 µg/ml in TBS/0.25% BSA for 1 h at room temperature. Plates were washed four times with TBS-T, followed by incubation with goat anti-human IgG F(ab’)₂ (Jackson ImmunoResearch) at 1:2000 dilution in TBS/0.25% BSA for 1 h at room temperature. Plates were washed six times in TBS-T and developed with Sure Blue Reserve TMB Microwell Peroxidase Substrate (KPL) for approximately 90 s. The reaction was stopped by addition of TMB Stop Solution, and absorbance at 450 nm was measured using an ELISA plate reader. Nonspecific binding of the human anti-tau mAbs was determined by measuring binding to non-peptide coated wells and subtracted for each antibody.

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.