9 1992 by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/92/3502-0153 $02.80

Development and Application of an -Face-Specific Radioimmunoassay for Vitamin D. GLENN KENNEDY,* W. JOHN BLANCHFLOWER, PAUL B. YOUNG, AND W. BRIAN DAVIDSON Biochemistry Section, Veterinary Sciences Division, Department of Agriculture for Northern Ireland, Stormont, Belfast BT4 3SD, Northern Ireland, UK Received November 19, 1991; Accepted February 4, 1992

ABSTRACT The first development of an o~-face-specific radioimmunoassay for vitamin B12 is described. Sheep, fed a cobalt-deficient diet, and immunized with a conjugate between Co-~ carboxypropyl cobalamin and keyhole limpet hemocyanin, were used to produce antisera. The antisera crossreacted with Co-~ derivatives of vitamin B12, but did not crossreact with the ~-face vitamin B]2 analog cobinamide. The antisera were used to develop a sensitive and reproducible radioimmunoassay that was free from contamination with the nonspecific vitamin B12 binding protein, R-protein. Both the radioimmunoassay and measurements of plasma concentrations of methylmalonic acid were applied to the diagnosis of cobalt/vitamin B12 deficiency in sheep. The assay correlated well with a commercially available radioassay and did not falsely detect normal vitamin B12 concentration in plasma samples containing elevated concentrations of methylmalonic acid. Index Entries: Cobalt; vitamin B12, methylmalonic acid; radioimmunoassay; sheep.

*Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research

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Kennedy et al.

INTRODUCTION Quantification of plasma concentrations of vitamin B12 has been a highly controversial area for many years. A wide variety of microbiological, radioisotopic, and radioimmunoassay techniques for its determination have already been described (1). One of the major criticisms of many vitamin B12 assays is that they are poorly reproducible (2). Part of this problem may result from interference by nonphysiologically active vitamin B12 analogs in the various assay systems. The vitamin B12 molecule consists of a corrin ring with a central cobalt atom. A variety of ligands may be attached to the cobalt atom either below (o~-face)or above (B-face) the plane of the corrin ring. Vitamin B12 derivatives with structural variants linked to the 13-face of the molecule include the coenzyme forms of the vitamin, namely 5'-deoxyadenosyl cobalamin and methyl cobalamin. In contrast, vitamin B12 analogs have structural variants linked to the o~-face of the molecule and may be metabolically inactive (3). This can create a problem in that some of these analogs can support the growth of the organisms used in microbiological assays (4) and can bind to the vitamin B12 binding proteins employed in radioassays (5) leading in both cases to erroneous results. Similarly, all of the radioimmunoassays for vitamin B12 that have been developed to date are specific for the 13-face of the molecule (6-8) and may thus also detect the biologically inactive o~-face analogs. A major criticism of vitamin B12 radioassays is that the highly specific vitamin B12 binding protein employed, intrinsic factor, is frequently heavily contaminated with the nonspecific vitamin B12 binding protein known as R-protein (3). Commercially available vitamin B12 radioassays have attempted to overcome this problem by including cobinamide, a nonphysiological oL-faceanalog of vitamin B12, in their reagents. Cobinamide, at saturating concentrations, is believed to block R-protein binding activity while leaving intrinsic factor binding activity unaltered. However, this approach has been criticized as being difficult to control and is not recommended (4). In a comprehensive review of vitamin B12 radioassays and radioimmunoassays, Pratt and Woldring (9) suggested a number of strategies that might lead to the production of different antivitamin B12 antisera of potential use in radioimmunoassays. Included among these were suggestions for novel 0~- and 13-face-specific antisera. An antiserum that is o>face specific is most likely to discriminate against the inactive o>face analogs of vitamin B12 and thus may be the most useful antiserum to use in a vitamin B12 assay. Given that antihapten antisera tend to be specific for the part of the hapten molecule that is furthest from the site of attachment to the carrier protein, it follows that, in order to produce an n-face-specific antiserum against vitamin B12, it is necessary to attach vitamin B12 to the protein via the 13-face. This poses a technical problem. The only possible site of attachment through the B-face is by forming a cobalt---carbon bond. However, 13-face cobalt--carbon bonds are very Biological Trace Element Research

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light sensitive, hydrolyzing very rapidly to form hydroxy cobalamin. Consequently, all chemical manipulations involving compounds with a 6-face cobalt--carbon bond must be performed under dim red illumination. In this laboratory, we have been studying the biochemistry of cobalt deficiency in sheep (10-12). Cobalt is an essential trace element in sheep, being required for the ruminal synthesis of vitamin B12. Being herbivores, this represents their sole vitamin B]2 source, and sheep that consume

Development and application of an alpha-face-specific radioimmunoassay for vitamin B12.

The first development of an alpha-face-specific radioimmunoassay for vitamin B12 is described. Sheep, fed a cobalt-deficient diet, and immunized with ...
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