Naar J, Branaa P, Chinain M, Pauillac S
Unité d'Océanographie Médicale, Institut de Recherches Médicales Louis Malardé, P.O Box 30, Papeete, Tahiti, French Polynesia.
Bioconjug Chem. 1999 Nov-Dec;10(6):1143-9. doi: 10.1021/bc990042g.
A minute amount (0.446 micromol) of cholesterol (Chol) was converted into an hemisuccinate derivative (Chol HS) using an excess of succinic anhydride. The optimal conditions for synthesis of Chol HS were explored by checkerboard experiments in which various succinic anhydride/Chol molar ratios ranging from 5:1 to 30:1 were assayed over a wide temperature range (50-85 degrees C) and for various incubation 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 bovine serum albumin (BSA) then to various proteins (casein, ovalbumin, and hemocyanins) 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 (epitope density) was achieved chromatographically by two methods according to a Chol standard curve established at 207 nm with linearity in the range 0-50 microg. These procedures involving an 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. The influence of the activated hapten/BSA molar ratio on the coupling efficiency was investigated by the direct method within the range 10:1 to 250:1. Using the optimal conditions determined for Chol HS synthesis (a molar 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 carrier stock concentration of 5 mg/mL), epitope density of the conjugates lied between 23 and 27. By reacting the same amount of activated hapten ( approximately 216 microg) with identical amounts of various carriers (300 microg), conjugation efficiency was found similar on a microgram of Chol bound per milligram of carrier basis. This simple and reproducible conjugation and analysis procedures should provide a general method applicable to poorly available and weakly immunogenic haptens bearing hydroxyl groups such as polyether-type marine toxins.
使用过量的琥珀酸酐将微量（0.446微摩尔）胆固醇（Chol）转化为半琥珀酸衍生物（Chol HS）。通过棋盘实验探索Chol HS的最佳合成条件，其中在较宽温度范围（50 - 85℃）和不同孵育时间（3 - 8小时）下测定了从5:1到30:1的各种琥珀酸酐/Chol摩尔比。在较高的试剂比例、温度和孵育时间下可实现完全转化。随后，这种羧酸衍生物首先通过在反胶束介质中进行的改良版Erlanger混合酸酐法与牛血清白蛋白（BSA）共价连接，然后与各种蛋白质（酪蛋白、卵清蛋白和血蓝蛋白）或合成均聚物（聚-DL-赖氨酸）连接。根据在207nm处建立的Chol标准曲线（线性范围为0 - 50微克），通过两种色谱方法评估每摩尔BSA的半抗原基团数量（表位密度）。这些程序包括对缀合物样品（直接法）或未反应的Chol HS（间接法）进行碱性水解，在所有情况下都产生了可接受的一致性水平和一致的结果。通过直接法在10:1至250:1的范围内研究了活化半抗原/BSA摩尔比对偶联效率的影响。使用为Chol HS合成确定的最佳条件（试剂摩尔比为30:1，在65℃孵育6小时）和BSA半抗原化条件（活化半抗原摩尔过量100倍，载体储备浓度为5mg/mL），缀合物的表位密度在23至27之间。通过使相同量的活化半抗原（约216微克）与相同量的各种载体（300微克）反应，发现以每毫克载体结合的Chol微克数为基础，偶联效率相似。这种简单且可重复的偶联和分析程序应提供一种适用于难以获得且免疫原性弱的带有羟基的半抗原（如聚醚型海洋毒素）的通用方法。
