Endocrine Abstracts

Obesity is a life-threatening, yet common condition that often lies at the root of other serious illnesses such as diabetes, cardiovascular disease and other neuroendocrine disorders. The central nervous system signalling pathways regulating food intake are complex and the precise mechanisms remain to be determined. Cocaine- and amphetamine- regulated transcript (CART) is a CNS neuropeptide involved in the regulation of feeding.
Development of a two-site immunometric assay requires antibodies to two spatially separated epitopes. It was anticipated that CART peptide (55-102) conjugated to BSA using conventional carbodiimide techniques would easily elicit the required spectrum of antibodies. However, this proved not to be the case.
The following series of antibodies were produced;
1. CART(55-102) Sheep Pc Ab
2. CART(55-102) Mab (clone G3/H12)
3. CART(79-102) Rabbit Pc Ab
4. Immune-complex (CART(55-102) bound to purified (1.)) Sheep Pc Ab
Each antibody combination was examined in the same format i.e Mab Label incubated with CART(55-102) standard prior to incubation with Sepharose solid-phase purified Pc Ab.
Results showed that the Mab used in combination with either the sheep or the Rabbit Pc Abs did not detect the CART(55-102) standard. However, success was achieved with the Mab label and the immune complex Sheep Pc Ab solid-phase combination.
Displacement studies suggested an immunodominant epitope within the CART(55-102) fragment. This may be due to the folded tertiary structure of the peptide, or because the antibody characterisation method uses 125I CART(55-102) iodinated via tyrosine residues. In the latter case, c-terminal antibodies would be expected to predominate.