ThT结合试验法检测Aβ42聚集情况的条件探讨

The study on conditions of ThT binding assay detecting Aβ42 aggregation

目的探讨硫黄素（ThT）结合试验法检测β-淀粉样多肽1-42（Aβ42聚集情况的条件。方法 Aβ42六氟异丙醇（HFIP）单体化处理后,进行复溶,然后将Aβ42ThT溶于缓冲体系,一定温度下于96孔板中共孵育不同时间,酶标仪检测450 nm激发下485 nm的荧光强度。分析Aβ42同的浓度,孵育温度,96孔板类型,缓冲液种类以及复溶方式对ThT结合试验法测定Aβ42集情况的影响。结果在ThT结合试验法中,Aβ42育浓度与荧光值成线性正比关系,孵育温度对此正比关系无较大影响;选用半透明板及PBS缓冲体系与其它条件相比,此线性正比关系更稳定;以最终NH3·H2O 20 mmol/L的浓度加入1%NH3·H2O对单体化处理后的Aβ42溶,并进行过滤的方法,与其它方法相比,寡聚体更少,以最终2%v/v（DMSO/PBS）的比例加入二甲基亚砜（DMSO）进行复溶的方法,寡聚体较多,且Aβ42快更多地发生了聚集;而利用ThT结合试验法研究Aβ42单体到聚合体进程变化的动力学时,使用20 mmol/L NH3·H2O复溶方式进行复溶,观察到的动力学趋势过程更明显直观。结论不同条件对ThT结合试验法检测Aβ42集情况的结果影响较大,条件不同结果不同,实验中应根据实验目的及具体情况来选择不同的条件。

Objective To investigate the conditions of ThT binding assay detecting β-amyloid1-42（Aβ42 aggregation.Methods Aβ42were subject to monomerization by hexafluoroisopropanol（HFIP）. Then monomeric Aβ42were redissolved and diluted in buffer with ThT together. Next,the solution was added into 96-well plates incubated for different time under a certain temperature. Under the excitation of 450 nm,we could detect their intensity of fluorescence at 485 nm emission to study the effects of Aβ42different concentrations,incubation temperatures,96-well plates types,buffer types and redissolving methods on ThT binding assay detecting Aβ42aggregation. Results In ThT binding assay,the relationship between Aβ42concentration and the fluorescence values showed linearly direct ratio which was hardly affected by incubation temperature. And the linear proportional relationship was more steady compared with others in terms of Costar 96 clear bottom black side and PBS buffer. The redissolving method of adding 1% NH3·H2O to monomerized Aβ42with a final concentration of 20 mmol/L NH3·H2O combined with filtration led to less oligomers compared with other methods. Another redissolving method of adding DMSO to monomeric Aβ42with a final concentration of 2% DMSO contributed to more oligomers compared with other methods. And this one was a much quicker way to form aggregates. Meanwhile,to investigate Aβ42kinetic change from monomer to polymer with ThT binding assay,choosing the redissolving method of 20 mol/LNH3·H2O was more likely to observe a dynamic trend. Conclusion Different conditions had influences on detecting Aβ42aggregation by ThT binding assay. Since different conditions had different results,choosing different experimental conditions should be based on the experiment purpose and specific situation.