Ap conjugated anti human igg secondary antibody

The binding of LN01, 10E8 and HK20 (Sabin et al., 2010 (link)) mAbs to the 28 amino acids long peptide encompassing the entire gp41 MPER was tested by ELISA using 96 well plates half-area. Briefly, ELISA plates were coated with 2 μg/ml of the long peptide (Pepscan), blocked with 1% BSA and incubated with titrated antibodies, followed by AP-conjugated anti-human IgG secondary antibody (SouthernBiotech). Plates were then washed and substrate (p-NPP, Sigma) was added. After 1h of incubation, plates were read at 405 nm. For human IgG quantification in the plasma of mice from in vivo experiment, 96 well plates were coated with 10 μg/ml of goat anti-human IgG UNL (SouthernBiotech), blocked with 1% BSA and incubated with titrated plasma, followed by AP-conjugated anti-human IgG secondary antibody (SouthernBiotech). Plates were then washed and substrate (p-NPP, Sigma) was added. After 30 min of incubation, plates were read at 405 nm. As standard to quantify human IgG concentration, serial dilutions of Rituximab were used.

Human mAbs and total IgG from PAP sera were purified by protein A or protein G chromatography (GE Healthcare). Total IgG from healthy donors were purified using the HiTrap Protein A HP columns (GE Healthcare) and concentrated using Amicon Ultra filter units (100 K, Millipore). Total GM-CSF antibodies were affinity-purified from PAP sera using magnetic beads (Invitrogen) conjugated with human GM-CSF. Total IgGs were quantified using ELISA plates coated with anti-human IgG (SouthernBiotech) using Certified Reference Material 470 (ERMs-DA470, Sigma-Aldrich) as standard. Binding to GM-CSF was tested by ELISA using 384-well SpectraPlates (PerkinElmer) for primary screenings or 96-well MaxiSorp plates (Nunc) for any following test. Briefly, ELISA plates were coated with 1 μg ml⁻¹ of recombinant human GM-CSF (Gentaur), blocked with 1% BSA and incubated with titrated antibodies, followed by AP-conjugated anti-human IgG secondary antibodies (SouthernBiotech). Plates were then washed, substrate (p-NPP, Sigma) was added and plates were read at 405 nm. EC50 (ng ml⁻¹) was calculated for every sample by nonlinear regression analysis using the GraphPad Prism 5 software.