An improved method for the microscale preparation and characterization of hapten-protein conjugates: The use of cholesterol as a model for nonchromophore hydroxylated haptens
An improved method for the microscale preparation and characterization of hapten-protein conjugates: The use of cholesterol as a model for nonchromophore hydroxylated haptens
复制标题
DOI：
10.1021/bc990042g
复制
发表时间：
1999-11-01
影响因子：
4.7
通讯作者：
Pauillac, S
中科院分区：
文献类型：
--
作者：
Naar, J;Branaa, P;Pauillac, S
使用过量的琥珀酸酐将微量（0.446 μ mol）的胆固醇（Chol）转化为半琥珀酸酯衍生物（Chol HS）。通过棋盘实验探索了合成Chol HS的最佳条件，在棋盘实验中，在宽的温度范围（50-85 ℃）和不同的孵育时间（3-8 h）内测定了琥珀酸酐/Chol摩尔比（5：1至30：1）。在较高的试剂比率、温度和孵育时间下获得总转化率。随后，该羧基衍生物首先共价连接到牛血清白蛋白（BSA），然后连接到各种蛋白质（酪蛋白、卵清蛋白和血蓝蛋白）或通过埃尔朗格的混合酸酐方法的修改版本（在反胶束介质中进行）连接到合成的均聚物（聚-DL-赖氨酸）。每摩尔BSA的半抗原基团数（表位密度）的评估通过两种方法根据在207 nm处建立的Chol标准曲线以色谱法实现，线性范围为0-50 μ g。这些程序涉及结合物（直接法）或未反应Chol HS（间接法）样品的碱性水解，在所有情况下均产生可接受的一致性水平和一致结果。通过直接法在10：1至250：1的范围内研究了活化的半抗原/BSA摩尔比对偶联效率的影响。使用为Chol HS合成（30：1的摩尔试剂比，在65 ℃下孵育6小时）和BSA半抗原化（100倍摩尔过量的活化半抗原，载体储备液浓度为5 mg/mL）确定的最佳条件，缀合物的表位密度在23和27之间。通过使相同量的活化半抗原（类似于216 μ g）与相同量的各种载体（300 μ g）反应，发现结合效率类似于每毫克载体结合的微克Chol。这种简单和可重复的缀合和分析程序应提供适用于难以获得的和弱免疫原性的带有羟基的半抗原如聚醚型海洋毒素的通用方法。
A minute amount (0.446 mu mol) 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 mu g. 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 (similar to 216 mu g) with identical amounts of various carriers (300 mu g), 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.