A quantitative bioassay for nerve growth factor, using PC12 clones expressing different levels of trkA receptors.
Nerve growth factor (NGF) is a neurotrophin required for differentiation, development, and survival of the sympathetic nervous system, with many of its biological effects being mediated via trkA receptors. There is a need for a standard quantitative bioassay for NGF, to be used in basic research and in pharmaceutical studies. The objective of the present research was to develop a selective, quantitative, and reliable bioassay for NGF, using a morphological criterion: neurite cell outgrowth. In addition, we aimed to apply the aforementioned bioassay to measure NGF administered to mice. Pheochromocytoma PC12 cell variants including wild-type cultures, and a trkA-overexpressing stable transfectant PC12-6.24-I, PC12nnr5, and PC12EN lacking trkA receptors, were used. Dose-response curves were generated with NGF beta-subunit (2.5S) purified from mouse submaxillary glands. Our results demonstrated that the bioassay was sensitive to 0.3-20 ng/mL, and selective, as neurite outgrowth was not seen by any other growth factor other than NGF. In addition, variant clones PC12nnr5 and PC12EN, lacking trkA receptors, did not respond to NGF. The bioassay detected NGF in serum of mice injected with NGF. This novel developed bioassay can serve as a model system for various neuroscience purposes.
Katzir
Itzhak
I
Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel.
Shani
Jashovam
J
Regev
Keren
K
Shabashov
Dalia
D
Lazarovici
Philip
P
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D015415:Biomarkers; D004305:Dose-Response Relationship, Drug; D051379:Mice; D020932:Nerve Growth Factor / Q000032:analysis* / Q000378:metabolism; D016501:Neurites / Q000378:metabolism; D016716:PC12 Cells; D011485:Protein Binding; D051381:Rats; D020917:Receptor, trkA / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015415:Biomarkers; D020932:Nerve Growth Factor; D020917:Receptor, trkA