ELISA 实验优化：十大关键因素全面解析

<p>&nbsp;<font face="sans-serif" style="color: rgb(68, 68, 68); font-family: sans-serif; font-size: 9.5pt; letter-spacing: 0.1pt;">酶联免疫吸附试验</font><span style="color: rgb(68, 68, 68); font-family: sans-serif; font-size: 9.5pt; letter-spacing: 0.1pt;"> (ELISA) 是一种广泛使用的实验室技术，可帮助科研人员检测样本中特定蛋白质的存在。优化 ELISA 实验条件对于确保结果准确可靠至关重要。本文将探讨优化 ELISA 实验条件的关键因素。</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <h3><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">抗原涂层</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></h3> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">ELISA的第一步是用目标抗原包被微孔板。包被抗原浓度的选择至关重要。抗原过少可能导致信号弱，而抗原过多则会导致非特异性结合。研究人员通常会进行滴定来确定最佳抗原浓度，以在不影响特异性的情况下提供强信号。</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <h3><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">阻塞</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></h3> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">抗原包被平板后，必须封闭任何剩余的结合位点，以防止非特异性相互作用。常见的封闭剂包括牛血清白蛋白</font><font face="sans-serif">(BSA)或脱脂奶粉。应优化封闭剂的浓度和选择，以最大限度地降低背景噪音，并最大限度地提高抗体与包被抗原的特异性结合。</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <h3><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">抗体浓度</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></h3> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">选择合适的一抗和二抗浓度对于灵敏度和特异性至关重要。抗体过少可能导致信号弱，而抗体过多则会增加背景噪音。通常进行滴定实验以确定能够提供最佳信噪比的最佳抗体浓度。</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <h3><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">孵化时间</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></h3> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">孵育步骤的持续时间是影响</font><font face="sans-serif">ELISA结果的另一个因素。抗体孵育时间过短可能导致信号弱，而孵育时间过长则会增加背景噪音。科研人员必须尝试不同的孵育时间，以确定每个步骤（包括抗原包被、封闭和抗体孵育）的最佳持续时间。</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">洗涤</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">孵育步骤之间彻底清洗对于去除未结合的抗体和其他成分至关重要。清洗不充分会导致背景噪音过大，并降低检测灵敏度。优化清洗次数和持续时间有助于确保背景清晰，并准确检测特定信号。</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <h3><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">底物和检测</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></h3> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">底物和检测系统的选择是</font><font face="sans-serif">ELISA优化的关键环节。常用的底物包括3,3',5,5'-四甲基联苯胺(TMB)或2,2'-连氮-双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)。检测系统（无论是比色、化学发光还是荧光）应根据所需的灵敏度和实验室现有的仪器进行选择。</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <h3><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">校准曲线</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></h3> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">为了准确定量目标蛋白的浓度，必须使用已知标准品生成校准曲线。优化标准曲线需要测试一系列浓度，以确定检测的线性范围和检测下限。这确保</font><font face="sans-serif">ELISA能够可靠地定量特定范围内的蛋白浓度。</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">板材一致性</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">使用一致的微孔板批次对于重现性至关重要。微孔板材质或涂层的差异会导致测定结果出现差异。科研人员应验证每批微孔板的性能，并尽可能在单个实验中使用同一生产批次的微孔板。</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <h3><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">温度和湿度</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></h3> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">环境条件（例如温度和湿度）会影响检测性能。务必在受控条件下进行实验，并监测任何可能影响结果的温度或湿度变化。这对于长时间孵育步骤尤其重要，因为在这些步骤中，保持稳定的条件至关重要。</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <h3><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">数据分析</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></h3> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">最后，正确的数据分析对于准确解读</font><font face="sans-serif">ELISA结果至关重要。研究人员应使用合适的软件或统计方法来分析信号强度、比较样本并确定目标蛋白的浓度。标准化的数据分析程序有助于确保实验的一致性和可靠性。</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p> <p class="MsoNormal"><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="sans-serif">总而言之，优化</font><font face="sans-serif">ELISA实验条件是一个细致的过程，需要仔细考虑各种因素。研究人员必须进行系统性实验，对从抗原包被到数据分析的每个参数进行精细调整。通过关注这些细节，可以提高ELISA检测的灵敏度、特异性和可重复性，最终获得可靠且有意义的研究结果。</font></span><span style="font-family: sans-serif; color: rgb(68, 68, 68); letter-spacing: 0.1pt; font-size: 9.5pt; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><o:p></o:p></span></p>