Enzyme immunoassay of [beta] cyclodextrin

The cyclodextrins represent a family of oligosaccha-rides of annular structure which may contain 6, 7 or 8 glucoside units ([beta], [beta] et [beta] cyclodextrins respectively). The orientation of the glucose residues gives the molecule a hydrophobic character at the centre of the ring while the exterior has hydrophilic properties (see figure below). The cyclodextrins therefore have inter-esting properties as regards the inclusion of hydro-phobic molecules although these properties are re-stricted by the size of the central cavity. The inclusion properties of [beta]-cyclodextrin ([beta]-CD) have resulted in various uses. In chemistry, these molecules have been used as a reactant in stereospecific syntheses or in the chromatographic separation of very similar compounds or optical isomers. In the agrifoodstuffs and cosmetics industries, these compounds are used to protect volatile or oxidation-sensitive substances such as flavourings or perfumes. Finally, they are useful in solubilising and stabilising the active com-pound in drug formulation.

Using an analogue of [beta]-CD in which a hydroxyl group is substituted by a primary amine, an enzyme immu-noassay has been developed, using sensitive and reproducible competition, for [beta]-CD. It is based on the use of a polyclonal antiserum obtained from rabbit and an enzyme tracer obtained by covalent binding of [beta]-CD to acetylcholinesterase from electric eel (CEA patent). The assay is specific to ;[beta]-cyclodextrin since a and [beta]-CD are recognised to a very small extent as is the linear analogue of [beta]-cyclodextrin (maltohep-taose). Specificity studies have been undertaken with various synthetic analogues. It has been observed that modification of all the hydroxyls in position 6 does not modify recognition by the antibodies, con-trary to that of the hydroxyls in position 2 or 3. The epitope would therefore appear to be located on this part of the molecule. Finally, derivatives of [beta]-CD with the lateral chain (phenylalanine, tyrosine, C5 or C12 hydrocarbonated chain) included in the cavity, are also recognised by the antibodies.

The immunoassay is therefore perfectly suited to pharmacological studies or when monitoring drug formulations using b-cyclodextrin. C.C.

Assay characteristics

Detection limit: 0.120 ng/ml
Range: 0,02 to 50 ng/ml
Specificity: see above
Reproductibility: SD < 9[percent] of 0.2 to 50 ng/ml
IC 50 [percent]: 0,72 ng/ml

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