Goat anti human igg fc

Total IgG secreted in B-cell culture supernatants was measured using a sandwich Enzyme-linked Immunosorbent Assay (ELISA). Briefly 96-well Maxisorp flat bottom plates (Nunc) were incubated overnight at 4°C with a goat anti-human IgG (AbDSerotec) diluted 1:1000 in coating buffer (Carbonate-Bicarbonate Buffer, pH 9.5). The following day, the coating buffer was removed, and plates were blocked with 2% non-fat milk solution in PBS, 0.05% Tween 20 (PBST) for 1 hour on an orbital shaker. Plates were then treated with either supernatants removed from B lymphocyte cultures or different concentrations of a human IgG control antibody (Sigma), from which the standard curve was derived. Plates were incubated for 2 hours at room temperature on an orbital shaker, and bound antibodies were detected with horseradish peroxidase-labelled goat anti-human IgG Fc-specific antibody diluted 1:1000 in PBST (Sigma) for 45 minutes at room temperature on an orbital shaker. After washing of unbound antibody, colour reaction was developed with o-Phenylenediamine dihydrochloride (OPD, Sigma) and OD was measured in an ELISA reader (BMG Labtech) at 492 nm (reference wavelength, 650 nm). Anti-folate receptor alpha IgG ELISA was performed as previously described. The lower detection levels of quantification were 3.125 and 0.625 ng/ml, respectively for 1:5 dilution and neat samples [15 (link), 16 (link)].

For quantification of DMAb in culture supernatants, NUNC 96-well MaxiSorp plates (Sigma; M9410-ICS) were coated with 5 µg ml⁻¹ goat anti-human IgG-Fc (Bethyl; A80-104A) diluted in 1 × PBS overnight at 4 °C. The following day, plates were washed 4 times with 0.05% PBS-T and were blocked with 5% non-fat dry milk in PBS for 1 h at room temperature (RT). Plates were washed and incubated with duplicate samples, diluted in 1% newborn calf serum (NCS) in 0.2% PBS-T for 1 h at RT. Plates were washed and incubated with 1:10000 HRP-conjugated goat anti-human IgG-Fc (Bethyl; A80-104P) diluted in 1% NCS in 0.2% PBS-T for 1 h at RT. Finally, washed plates were developed with 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo; 34028) and quenched with 2N H₂SO₄. Plates were read at 450 nm on the BioTek Synergy 2 (Biotek) plate reader. Blank wells were included on each plate and subtracted as background. Purified human IgG (Bethyl; P80-112) was used to create a standard curve for quantification (µg ml⁻¹). Positive control sample was included on each plate and used to standardize values across assays. Data were subsequently exported to Microsoft Excel and analyzed using GraphPad Prism 9. Negative OD values (following background correction) were represented by zero for graphing purposes.

96-well immuno-sorbent plates (Nunc MaxiSorp, Thermo Fisher Scientific) were coated with 100 μl of goat anti-human IgG-Fc (Sigma-Aldrich) diluted to 1 μg/ml in 1x Dulbecco's phosphate-buffered saline (DPBS) (Gibco, Life Technologies) and were incubated overnight at 4°C without shaking. Next day wells were blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich) in 1x DPBS after a washing step. 1x DPBS containing 0.05% Tween 20 (Sigma-Aldrich) was used as washing buffer. After a further washing step, standard (Fc from human plasma, Calbiochem) and samples were added to the blocked microwells and incubated for one hour. As detection antibody horseradish peroxidase conjugated protein A (ProtA-HRP) (Sigma-Aldrich) diluted 1:80 000 in 2% BSA in 1x DPBS was used. As a colorigenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was used (Sigma-Aldrich). The reaction was stopped after 20 minutes by addition of 30% H₂SO₄ and absorbance was measured at 450 nm (620 nm reference wavelength) in a microplate reader (TECAN Infinite 200 Pro). A sigmoidal curve from standard was calculated and plotted with GraphPad Prism5.