Quantitative immunobinding assay for calbindin D/sub 28k/ using nitrocellulose filters

Conference · Sat Mar 01 04:00:00 EST 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5450753

Varghese, S; Chapman, E S; Christakos, S

An immunobinding assay has been developed for the quantitative determination of vitamin D dependent calcium binding protein (calbindin D/sub 28k/) from rat kidney and cerebellum. The protein samples are applied to nitrocellulose membrane filters using a slot-blot apparatus. The remaining protein binding sites are blocked with bovine serum albumin and incubated sequentially with rat renal calbindin D (CaBP) antiserum (1:500 dilution) and /sup 125/I protein A (200,000 cpm/ml). After washing, the radioactivity bound to each sample is quantitated by cutting the nitrocellulose sheets and counting in a gamma counter. Different dilutions of purified rat renal CaBP result in standard curves which are linear over a 40 to 50 fold range. The sensitivity of the assay is such that 10 ng of calbindin D can be accurately quantitated. The assay is precise (intraassay variability 6%) and reproducible (interassay variability 11.9%). Using this assay, the concentration of rat renal CaBP was found to be 8.8 +/- 1.3 ..mu..g/mg protein and the concentration of rat cerebellar CaBP was 34.0 +/- 6.0 ..mu..g/mg protein. There was good agreement between the data in this assay and the data which the authors previously reported using radioimmunoassay. Some advantages of this assay are that it is fast, reproducible and large numbers of samples can be handled in parallel. The most considerable advantage is that it does not require iodination of the antigen.

• Research Organization: UMDNJ-NJ Medical School, Newark
• OSTI ID: 5450753
• Report Number(s): CONF-8604222-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:3
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METALS
ANIMALS
BETA DECAY RADIOISOTOPES
BIOASSAY
BIOCHEMICAL REACTION KINETICS
BODY
BRAIN
CALCIUM
CARBOHYDRATES
CELLULOSE ESTERS
CENTRAL NERVOUS SYSTEM
CEREBELLUM
CHEMICAL EXPLOSIVES
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ELEMENTS
ESTERS
EXPLOSIVES
FILTERS
IMMUNE REACTIONS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KIDNEYS
KINETICS
MAMMALS
MEMBRANE PROTEINS
METALS
NERVOUS SYSTEM
NITRIC ACID ESTERS
NITROCELLULOSE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
POLYSACCHARIDES
PROTEINS
RADIOISOTOPES
RATS
REACTION KINETICS
RECEPTORS
RODENTS
SACCHARIDES
TRACER TECHNIQUES
VERTEBRATES
VITAMIN D
VITAMINS