Production and characterization of polyclonal antibodies against cholecalciferol (vitamin D3)

Vitamin D is one of the essential vitamins in the human diet for normal growth and function. In Canada and the USA, fortified milk and milk products are the essential source of vitamin D. The adult recommended nutrient intake of vitamin D is 200 to 400 I.U. (corresponding to 5 to 10 microg) per day. Additional amounts of vitamin D do not confer benefits and may even be toxic. However, a deficiency of this vitamin leads to inadequate absorption of calcium and phosphorus and faulty mineralization of bones and teeth. Actual methods for measuring vitamin D in milk are limited in terms of sensitivity, rapidity and simplicity. The objective of this manuscript was to develop a new molecular strategy for the production, purification and characterization of polyclonal antibodies to vitamin D. Specific antibodies were raised in rabbits against vitamin D using cationized bovine serum albumin (cBSA) as a carrier protein. Anti-vitamin D antibodies were recovered from rabbit sera by sequential affinity chromatographies through Protein A/G Agarose, cBSA Sepharose and cOVA-vitamin D Sepharose columns. Although the yields of anti-vitamin D were relatively low, recovered antibodies showed high specificity and affinity to vitamin D. The purified antibody was used to develop a solid-phase enzyme immunoassay in order to determine the exact concentration of vitamin D in phosphate buffer. Using this immunoassay, approximately 35 ng of vitamin D can be detected within 3 h. The signal obtained was proportional to the amount of vitamin D in the sample analyzed. The strategy developed in this paper appears to be very promising in terms of sensitivity, rapidity and simplicity. It offers a great potential for automation and use on a routine basis for the quantification of vitamin D in fortified milk and other milk products.