Horseradish peroxidase hrp conjugated donkey anti rabbit igg

ELISA strips were coated with 3D9 (murine mAb to CR1; 100 μl at 2.5 μg/ml in PBS overnight at 4 °C) (Krych et al., 1991 (link)). Wells were blocked (1% BSA, 0.1% Tween-20, 0.02% sodium azide in PBS) for 2 h at 37 °C and washed in PBS with 0.05% Tween-20 (wash buffer). Dilution of cell lysates of the CHO transfectants (100 l) was made in sample buffer (PBS with 0.05% Tween-20, 0.25% non-ionic detergent Nonidet P-40, 4% BSA) and incubated for 1 h at 37 °C followed by three 2-min washes in wash buffer. Rabbit polyclonal anti-CR1 Ab (1:20,000 dilution in sample buffer) was incubated for 1 h at 37 °C followed by a similar washing schedule. Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (100 μl of a 1:15,000 dilution; GE healthcare, UK) was added and then incubated for 1 h at 37 °C followed by washing. Detection was made using orthophenylenediamine dihydrochloride (100 l of a 1:20 dilution) in a buffer containing 0.02% hydrogen peroxide in citrate phosphate buffer (pH 6.35). The reaction was stopped by adding 100 l of 2N sulfuric acid (2N H₂SO₄). The ELISA plates were read at 490 nm using the Micro Quant reader (Biotek Instruments, Inc). Soluble CR1 was used as the standard (gift from Henry Marsh, Celldex Therapeutics, Needham, MA) (Nickells et al., 1998 (link)).