Protein g agarose resin

IgG from monkeys were purified from the serum with Protein G agarose resin (Sigma Aldrich). The flow-through was used to purify IgA with Jacalin agarose resin (Thermo Fisher Scientific). ELISA was performed with 10¹⁰ phage particles/50 μL of PBS coated onto 96-well plates ON at 4°C (Nunc MaxiSorp flat bottom, ThermoFisher Scientific). For this assay, phage titration was performed by quantitative qPCR with fUSE primers (fUSE5 forward, as follows: 5′-TGAGGTGGTATCGGCAATGA-3′ and fUSE5 reverse: 5′-GGATGCTGTATTTAGGCCGTTT-3′). ELISA was also performed with 96-well plates coated with the synthetic peptide (10 μg/mL) CAKSMGDIVC or an unrelated synthetic control peptide (sequence CGRRAGGSC unless otherwise specified)^{Ref.21 (link)} ON at 4°C. Coated plates were blocked with PBS containing 5% low-fat milk and 1% BSA for 1 h at 37°C. Two-fold serial dilutions (starting at 1:4) of purified IgG or IgA were applied to the wells and incubated for 2 h at 37°C. Following three washes with PBS and PBS containing 0.1% of Tween 20 (PBST), bound antibodies were detected with an anti-monkey IgG (KPL; 074-11-021) or IgA (KPL: 074-11-011) HRP-conjugated. Purified polyclonal IgG anti-CAKSMGDIVC antibodies (Biomatik USA, Delaware) and anti-fd bacteriophage antibody served as positive controls. Plates were read at an absorbance of 450 nm.