Neutralization
: Anti-Nerve Growth Factor neutralizes the bioactivity of recombinant rat NGF, recombinant human NGF, and recombinant mouse NGF.
To measure this biological activity,18 recombinant rat NGF is incubated with various concentrations of the antibody for 1 hour at room temperature in a 96 well plate. Following this preincubation, human erythroleukemic cells, TF -1, are added to the wells.
The assay mixture, in a total volume of 100 l, containing antibody at concentrations from 0.001 to 1 g/ml,
recombinant rat NGF (4 g/ml), and TF-1 cells (1.0 -105cells/ml), is incubated at 37 C in a humidified CO2 chamber for 48 hours.
H-thymidine is added during the last 4 hours of the incubation. The cells are harvested onto glass fiber filters and the
H-thymidine incorporated into the DNA is measured.
The Neutralization Dose 50 (ND 50 ) is defined as that concentration required to yield one-half maximal inhibition of the cytokine activity on a responsive cell line, when that cytokine is present at a concentration just high enough to elicit a maximum response.
The Neutralization Dose 50 (ND 50 ) for Anti-Nerve Growth Factor - is approximately 0.02-0.1 g/ml in the presence of 10.0 g/ml of recombinant rat NGF using the human erythroleukemic TF-1 cell line.
The exact concentration of antibody required to neutralize recombinant rat NGF activity is dependent on the cytokine concentration, cell type, growth conditions, and the type of activity.