Application of a nitrocellulose immunoassay for quantitation of proteins secreted in culture media

A macro-dot immunoassay was developed to quantitate proteins (antigens) secreted in the culture media of primary rat hepatocytes. Dilutions of protein standards and undiluted spent culture media were applied to numbered sheets of nitrocellulose (NC) paper by vacuum filtration (in volumes up to 1 ml) through a specially designed macrofiltration apparatus constructed of plexiglass. Sequential incubation of the NC with bovine serum albumin blocking buffer, monospecific antibody, and ¹²⁵I Protein A enabled quantitation of protein concentration by determination of NC bound radioactivity. Linear and reproducible standard curves were obtained with fibrinogen, albumin, transferrin, and haptoglobin. A high degree of coefficient of correlation between radioactivity (cpm) and protein concentration was found. Intra- and intertest reproducibility was excellent (C.V.'s <7%). By using monospecific antibodies, single proteins (i.e., fibrinogen), as low as 32 ng/ml, could be quantified in heterogenous protein mixtures and in spent culture media. The assay was sensitive to the difference of fibrinogen secretion under nonstimulatory (serum-free hormonally defined medium, SFHD) and stimulatory (SFHD plus hydrocortisone) culture conditions. The procedure and techniques described are applicable to the quantitation of any protein in a suitable buffer.