Quantitative immunobinding assay for vitamin D-dependent calcium-binding protein (calbindin-D28k) using nitrocellulose filters

Journal Article · Sat Aug 15 04:00:00 EDT 1987 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(87)90218-1· OSTI ID:5520643

Varghese, S; Christakos, S

A sensitive dot immunobinding assay has been developed for the quantitative determination of vitamin D-dependent calcium-binding protein (calbindin-D28k; CaBP) in rat and human kidney and brain. Protein samples are spotted onto nitrocellulose sheets, fixed, and then rinsed with Tris-buffered saline. The remaining protein binding sites are blocked with bovine serum albumin, gelatin, or nonfat dry milk protein and the filters are then incubated sequentially with antiserum to calbindin-D28k (1:500 dilution) and /sup 125/I-protein A (200,000 cpm/ml). After washing, the radioactivity bound to each sample is quantitated by counting in a gamma counter. The sensitivity of the assay is such that 10 ng calbindin-D28k can be accurately quantitated. The highest levels of CaBP were detected in kidney (7.8 +/- 0.5 micrograms/mg protein) and cerebellum (22.1 +/- 1.4 micrograms/mg protein). Ten micrograms calmodulin, lactalbumin, or parvalbumin and 100 micrograms liver extract showed no reactivity in the assay. The assay is precise (intraassay variability, 4.0%) and reproducible (interassay variability, 8.8%). There was good agreement between the data in this assay and the data we obtained using radioimmunoassay (RIA). The assay has several advantages over the RIA. Iodination of pure antigen is not required and it is possible to detect membrane-bound and insoluble antigens using this assay. Also, the antiserum and /sup 125/I-protein A solutions can be saved and reused. This assay represents a major modification of the original immunobinding assays which used the less sensitive peroxidase stain. It is also an improvement over previous /sup 125/I immunobinding assays which were not quantitative but were used as antigen spot tests or which required iodination of the antibody.

• Research Organization: Univ. of Medicine and Dentistry of New Jersey, Newark
• OSTI ID: 5520643
• Journal Information: Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 165:1; ISSN ANBCA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BETA DECAY RADIOISOTOPES
BODY
BRAIN
CARBOHYDRATES
CELLULOSE ESTERS
CENTRAL NERVOUS SYSTEM
CHEMICAL EXPLOSIVES
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ESTERS
EXPLOSIVES
FILTRATION
IMMUNOASSAY
IMMUNOLOGY
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KIDNEYS
MAMMALS
MAN
NERVOUS SYSTEM
NITRIC ACID ESTERS
NITROCELLULOSE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
POLYSACCHARIDES
PREGNANCY
PRIMATES
PROTEINS
RADIOASSAY
RADIOIMMUNOASSAY
RADIOIMMUNOLOGY
RADIOISOTOPES
RATS
RODENTS
SACCHARIDES
SENSITIVITY ANALYSIS
SEPARATION PROCESSES
TRACER TECHNIQUES
VERTEBRATES
VITAMIN D
VITAMINS