Total IgGs were quantified using 96-well MaxiSorp plates (Nunc) coated with goat
anti-human IgG (SouthernBiotech, 2040-01) using Certified Reference Material 470
(ERMs-DA470, Sigma-Aldrich) as a standard. To test specific binding of antibody
constructs, ELISA plates were coated with 2 μg ml−1 of type I
recombinant human collagen (Millipore, CC050), 2 μg ml−1 of an
anti-human LAIR1 antibody (clone DX26, BD Biosciences 550810), 1 μg
ml−1 of recombinant human GM-CSF (Gentaur), 2 μg
ml−1 of an anti-PD1 or an anti-SLAM antibody (R&D Systems,
AF1086 and AF164). Plates were blocked with 1% bovine serum albumin (BSA) and incubated
with titrated antibodies, followed by AP-conjugated goat anti-human IgG, Fcγ
fragment specific (Jackson Immuno Research, 109-056-098). Plates were then washed,
substrate (p-NPP, Sigma) was added and plates were read at 405 nm.
anti-human IgG (SouthernBiotech, 2040-01) using Certified Reference Material 470
(ERMs-DA470, Sigma-Aldrich) as a standard. To test specific binding of antibody
constructs, ELISA plates were coated with 2 μg ml−1 of type I
recombinant human collagen (Millipore, CC050), 2 μg ml−1 of an
anti-human LAIR1 antibody (clone DX26, BD Biosciences 550810), 1 μg
ml−1 of recombinant human GM-CSF (Gentaur), 2 μg
ml−1 of an anti-PD1 or an anti-SLAM antibody (R&D Systems,
AF1086 and AF164). Plates were blocked with 1% bovine serum albumin (BSA) and incubated
with titrated antibodies, followed by AP-conjugated goat anti-human IgG, Fcγ
fragment specific (Jackson Immuno Research, 109-056-098). Plates were then washed,
substrate (p-NPP, Sigma) was added and plates were read at 405 nm.