| Abstract
| - A minute amount (0.446 μmol) of cholesterol (Chol) was converted into an hemisuccinate derivative(Chol HS) using an excess of succinic anhydride. The optimal conditions for synthesis of Chol HSwere explored by checkerboard experiments in which various succinic anhydride/Chol molar ratiosranging from 5:1 to 30:1 were assayed over a wide temperature range (50−85 °C) and for variousincubation times (3−8 h). Total conversion was obtained at the higher reagent ratios, temperatures,and incubation times. Subsequently, this carboxylic derivative was first covalently linked to bovineserum albumin (BSA) then to various proteins (casein, ovalbumin, and hemocyanins) or to a synthetichomopolymer (poly-dl-Lysine) via a modified version of the mixed anhydride method of Erlanger,performed in a reversed micellar medium. The assessment of the number of haptenic groups per moleof BSA (epitope density) was achieved chromatographically by two methods according to a Cholstandard curve established at 207 nm with linearity in the range 0−50 μg. These procedures involvingan alkaline hydrolysis of a sample of either the conjugate (direct method) or the unreacted Chol HS(indirect method) yielded an acceptable level of agreement and concordant results in all cases. Theinfluence of the activated hapten/BSA molar ratio on the coupling efficiency was investigated by thedirect method within the range 10:1 to 250:1. Using the optimal conditions determined for Chol HSsynthesis (a molar reagent ratio of 30:1 with incubation at 65 °C for 6 h) and for BSA haptenation (a100-fold molar excess of activated hapten, with a carrier stock concentration of 5 mg/mL), epitopedensity of the conjugates lied between 23 and 27. By reacting the same amount of activated hapten(∼216 μg) with identical amounts of various carriers (300 μg), conjugation efficiency was found similaron a microgram of Chol bound per milligram of carrier basis. This simple and reproducible conjugationand analysis procedures should provide a general method applicable to poorly available and weaklyimmunogenic haptens bearing hydroxyl groups such as polyether-type marine toxins.
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