Polysaccharide-Specific Sandwich ELISA for PnPs

A polysaccharide-specific Sandwich ELISA was employed to screen the anti C-Ps mAb against several PnPs serotypes as described previously [31 (link)]. In this ELISA assay, the plates were coated with a CRM capture mAb (1 μg/mL). At 2–8 °C, the coated plates were blocked and incubated overnight with serially diluted target PnPs serotypes. After the plate wash, the anti C-Ps mAb (1 μg/mL) was applied to the plates and they were incubated for one hour. The plates were washed, then incubated for one hour with 1:6000 dilution of donkey anti-human antibody conjugated with alkaline phosphatase (Jackson ImmunoResearch, West Grove, PA, USA) in a solution of assay diluent spiked with normal mouse serum (Jackson ImmunoResearch). The plates were washed, 4-Methylumbelliferyl-phosphate (4-MUP) (Virolabs, Chantilly, VA, USA) was applied, and they were incubated for 45 min. The fluorescence signal was recorded on a SpectraMax M2E spectrophotometer at 360 nm (excitation)/450 nm (emission). Data were collected using SoftMax Pro (Molecular Devices, San Jose, CA, USA).