Sensitive enzyme immunoassay for human 28 kDa calbindin-D

Area of Science:

• Biochemistry
• Immunology
• Molecular Biology

Background:

• Calbindin-D is a 28 kDa vitamin D-dependent calcium binding protein.
• Understanding calbindin-D distribution is crucial for various physiological processes.

Purpose of the Study:

• To develop a sensitive and specific sandwich-type enzyme immunoassay for human 28 kDa calbindin-D.
• To characterize the distribution of calbindin-D in human tissues and body fluids.

Main Methods:

• Generation of antisera in rabbits using purified human kidney calbindin-D.
• Purification of specific antibodies and their labeling with beta-D-galactosidase.
• Development of a sandwich immunoassay using immobilized antibodies on polystyrene balls.

Main Results:

• The assay demonstrated high sensitivity (1 pg/tube) and specificity, with no cross-reactivity to other calcium binding proteins.
• Reproducibility was confirmed with inter-assay coefficients of variation below 10%.
• Significant calbindin-D concentrations were detected in human kidney and brain, with measurable levels in urine and cerebrospinal fluid.

Conclusions:

• The developed enzyme immunoassay is a reliable tool for quantifying human calbindin-D.
• Calbindin-D is widely distributed in human tissues and body fluids, with highest concentrations in the kidney.
• Immunohistochemical analysis localized calbindin-D within the epithelial cells of distal tubules in the human kidney.