Development and validation of an assay to measure bioactivity of human calcitonin in vitro using T47D cell membranes.
We have developed a "test-tube" assay to determine the biological activity of synthetic preparations of human calcitonin (hCT). The method is based on the dose-dependent accumulation of cyclic AMP in cell membrane preparations on stimulation with CT. As an unlimited cell source, we used the clonal cell line T47D, which possesses functional CT receptor-adenylate cyclase complexes. Half-maximal stimulation was achieved with 40-200 nmol/liter hCT. The method was able to precisely quantify the relative potencies of various hCT preparations. The intraassay and interassay coefficients of variation were 3.0 and 5.6%, respectively. The analytical performance, as estimated by additional recovery and specificity studies, was better than or comparable to the established in vivo rat hypocalcemia assay. Results obtained with these two methods were closely correlated (r = 0.83, P < 0.01). However, the in vitro membrane system allowed a higher throughput of samples, less preparation time, and better standardization and transportability of the assay, as large-scale membrane preparations were stable for at least 12 months in liquid nitrogen. This new in vitro membrane bioassay provides a convenient alternative to the currently performed in vivo rat hypocalcemia bioassays.
Department of Internal Medicine I, Endocrinology and Metabolism, University of Heidelberg, Germany.
E
E
Blind
F
F
Raue
P
P
Kienle
J
J
Schroth
A
A
Grauer
A
A
Kabay
P
P
Brügger
R
R
Ziegler
D000818:Animals; D001681:Biological Assay / Q000379:methods* / Q000706:statistics & numerical data; D002116:Calcitonin / Q000032:analysis* / Q000494:pharmacology; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D006996:Hypocalcemia / Q000139:chemically induced; D066298:In Vitro Techniques; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D002116:Calcitonin; D000242:Cyclic AMP