Application of a nitrocellulose immunoassay for quantitation of proteins secreted in cultured 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 plexiglas. Sequential incubation of the NC with bovine serum albumin blocking buffer, monospecific antibody, and /sup 125/I 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 (cmp) and protein concentration was found. Intra- and inter-test reproducibility was excellent. By using monospecific antibodies, single proteins (i.e., fibrinogen), as low as 32 ng/ml, could be quantified in heterogeneous protein mixtures and in spent culture media. The assay was sensitive to the difference of fibrinogen secretion under nonstimulatory (serum-free hormonally define 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.
- Research Organization:
- Johns Hopkins Univ. School of Medicine, Baltimore, MD
- OSTI ID:
- 5993639
- Journal Information:
- Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 158:2; ISSN ANBCA
- Country of Publication:
- United States
- Language:
- English
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62 RADIOLOGY AND NUCLEAR MEDICINE
ALBUMINS
ANIMAL CELLS
ANIMALS
ANTIBODIES
ANTIGENS
BETA DECAY RADIOISOTOPES
BLOOD COAGULATION FACTORS
CARBOHYDRATES
CELLULOSE ESTERS
CHEMICAL ANALYSIS
CHEMICAL EXPLOSIVES
COAGULANTS
CULTURE MEDIA
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ESTERS
EXPLOSIVES
FIBRINOGEN
GLOBULINS
GLOBULINS-BETA
HEMATOLOGIC AGENTS
HEMOSTATICS
IMMUNOASSAY
IMMUNOLOGY
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LIVER CELLS
MAMMALS
METALLOPROTEINS
NITRIC ACID ESTERS
NITROCELLULOSE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
POLYSACCHARIDES
PROTEINS
QUANTITATIVE CHEMICAL ANALYSIS
RADIOASSAY
RADIOCHEMICAL ANALYSIS
RADIOIMMUNOASSAY
RADIOIMMUNOLOGY
RADIOISOTOPES
RATS
RODENTS
SACCHARIDES
SECRETION
SOMATIC CELLS
TRACER TECHNIQUES
TRANSFERRIN
VERTEBRATES