Chemikine ngf sandwich elisa

Following incubation in NGF solution, loading and release of growth factor was evaluated under infinite sink conditions in vitro at 37 °C. NGF release was measured for 21 days following initial treatment. Allografts were washed three times in the first 24 hours and once every 24 hours subsequently with 1ml of Tris-buffered saline per wash. The washes were collected and stored in silanized Eppendorf tubes at −20 °C. After 21 days, the nerves were homogenized and total protein was extracted and quantified as before. Concentrations of NGF in nerve lysates and collected washes were then quantified in triplicate using an NGF sandwich ELISA (ChemiKine NGF Sandwich ELISA, Millipore). Washes from day 7 were retained for use in the bioactivity assay.

NGF-β (>98% pure as determined by RP-HPLC and SDS-PAGE; ProSpec, East Brunswick, NJ) was reconstituted to 100 μg/ml in sterile 18MΩ-cm H2O and filtered through a 0.22 μm filter. Processed nerve grafts were incubated in NGF-β solution for 16 hours at room temperature under agitation. Concentration of incubation solution was initially evaluated in a dilution series with Freeze-thaw processed nerve grafts and sandwich ELISA (ChemiKine NGF Sandwich ELISA, Millipore, Billerica, MA) of tissue lysates. Optimal incubation concentration of 10 μg/ml was chosen at the saturation point of NGF loading of tissue samples.