An improved method for the microscale preparation and characterization of hapten-protein conjugates: The use of cholesterol as a model for nonchromophore hydroxylated haptens

A minute amount (0.446 mu mol) of cholesterol (Chol) was converted into an hemisuccinate derivative (Chol HS) using an excess of succinic anhydride. T he optimal conditions for synthesis of Chol HS were explored by checkerboar d experiments in which various: succinic anhydride/Chol molar ratios rangin g from 5:1 to 30:1 were assayed over a wide temperature range (50-85 degree s C) and for various incubation times (3-8 h). Total conversion was obtaine d at the higher reagent ratios, temperatures, and incubation times. Subsequ ently, this carboxylic derivative was first covalently linked to bovine ser um albumin (BSA) then to various proteins (casein,: ovalbumin, and hemocyan ins) or to a synthetic homopolymer (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 mole of BSA (e pitope density) was achieved chromatographically by two methods according t o a Chol standard curve established at 207 nm with linearity in the range 0 -50 mu g. These procedures involving an alkaline hydrolysis of a sample of either the conjugate (direct method) or the unreacted Chol HS (indirect met hod) yielded an acceptable level of agreement and concordant results in all cases. The influence of the activated hapten/BSA molar ratio on the coupli ng efficiency was investigated by the direct method within the range 10:1 t o 250:1. Using the optimal conditions determined for Chol HS synthesis (a m olar reagent ratio of 30:1 with incubation at 65 degrees C for 6 h) and for BSA haptenation (a 100-fold molar excess of activated hapten, with a carri er stock concentration of 5 mg/mL), epitope density of the conjugates lied between 23 and 27. By reacting the same amount of activated hapten (similar to 216 mu g) with identical amounts of various carriers (300 mu g), conjug ation efficiency was found similar on a microgram of Chol bound per milligr am of carrier basis. This simple and reproducible conjugation and analysis procedures should provide a general method applicable to poorly available a nd weakly immunogenic haptens bearing hydroxyl groups such as polyether-typ e marine toxins.