Production and characterization of polyclonal antibodies against cholecalciferol (vitamin D-3)

Vitamin D is one of the essential vitamins in the human diet for normal gro wth and function. In Canada and the USA, fortified milk and milk products a re 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 mu g) per day. A dditional amounts of vitamin D do not confer benefits and may even be toxic . However, a deficiency of this vitamin leads to inadequate absorption of c alcium 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 characterizat ion of polyclonal antibodies to vitamin D. Specific antibodies were raised in rabbits against vitamin D using cationized bovine serum albumin (cBSA) a s a carrier protein. Anti-vitamin D antibodies were recovered from rabbit s era by sequential affinity chromatographies through Protein A/G Agarose, cB SA Sepharose and cOVA-vitamin D Sepharose columns. Although the yields of a nti-vitamin D were relatively low, recovered antibodies showed high specifi city and affinity to vitamin D. The purified antibody was used to develop a solid-phase enzyme immunoassay in order to determine the exact concentrati on 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 prop ortional to the amount of vitamin D in the sample analyzed. The strategy de veloped in this paper appears to be very promising in terms of sensitivity, rapidity and simplicity. It offers a great potential for automation and us e on a routine basis for the quantification of vitamin D in fortified milk and other milk products. (C) 1999 Elsevier Science B.V. Al rights reserved.