75% ultra-low IgG fetal bovine serum (both from Invitrogen)
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75% ultra-low IgG fetal bovine serum (both from Invitrogen).

MAbs were purified by means of affinity chromatography using a HiTrap Protein G HP column (GE Healthcare, Piscataway, NJ USA) under previously described conditions (Akerstrom and Bjorck, 1986). MAbs were biotinylated using biotin N-hydroxysuccinimide ester according to the manufacturer’s recommendations. Briefly, purified antibodies were dialyzed against 0.1 M carbonate buffer, pH 8.5, and biotin N-hydroxysuccinimide ester was added corresponding to 1/6 (w/w) of the total protein amount. The mixture was incubated with gentle mixing for 4 h at room temperature. Unreacted biotinylation reagent was removed by dialysis against TBS, pH 7.4. Proteins were separated by SDS-PAGE in 4–12% Novex Bis-Tris gels (Invitrogen) and blotted onto polyvinylidene difluoride membranes (PVDF HyBond, Amersham Biosciences, learn more Little Chalfont, UK). The membranes were washed and blocked for 1 h in washing buffer, followed by overnight incubation at 4 °C with biotinylated primary MAbs (0.52 μg/ml of 11–2, 1.0 μg/ml of 14–29 or 1.0 μg/ml of isotype-matched (IgG1κ) control anti-mouse SP-D K403) diluted in washing buffer. After repeated washing, the membranes were incubated for 1 h at room temperature in horseradish peroxidase (HRP)-conjugated Streptavidin (Invitrogen) diluted to 1/10,000 in washing buffer. The membranes GSK-3 signaling pathway were washed and developed with aminoethyl

carbazol. Polystyrene microwell plates (Maxisorp, Nunc, Roskilde, Denmark) were coated with 5 μg MAb 11–2 per ml coating buffer (100 μl/well). After overnight incubation at 4 °C, the coated wells were washed three times and left to block with washing buffer for 30 min at room temperature. The calibrator, controls and samples were diluted

in washing buffer containing bovine serum (0.1% v/v; AH diagnostics, Aarhus, Denmark) and heat-aggregated human IgG (50 μg/ml; Innovative Research, Ureohydrolase Novi, MI, USA) and incubated overnight at 4 °C. Subsequently, the wells were washed three times and biotinylated MAb 14–29 diluted to 0.5 μg/ml in washing buffer containing BSA (1 mg/ml) was added to the wells and incubated for 1 h at room temperature. After three washes, HRP-conjugated Streptavidin diluted to 1/12,000 in washing buffer containing BSA (1 mg/ml) was added to the wells and incubated for 30 min at room temperature. The wells were washed three times and 0.4 mg of o-phenylene-diamine (Kem-En-Tec, Taastrup, Denmark) was added per ml substrate buffer. After 15 min the color development was stopped with 1 M H2SO4. Optical density (OD) was measured at 490–650 nm using Vmax Kinetic Microplate Reader and the data were processed using SoftMax Pro software (Molecular Devices, Wokingham, United Kingdom). The samples were diluted to 1/40 and the calibrator, quality controls (QCs) and samples were run in triplicates unless otherwise stated.

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