细胞培养上清液ELISA检测的样品制备方法

<p>&nbsp;<span style="font-family: 宋体; font-size: 10.5pt;">&nbsp; &nbsp;&nbsp;</span><font face="宋体" style="font-family: 宋体; font-size: 10.5pt;">对于细胞样品，可根据目标物的位置选择细胞裂解液或细胞培养上清液作为样品。由于细胞样品存在诸多干扰因素，例如细胞状态、细胞数量、</font><font face="Calibri" style="font-size: 10.5pt;">pH</font><font face="宋体" style="font-family: 宋体; font-size: 10.5pt;">值、培养基盐离子浓度、采样时间等，可能会出现样品无法检测的情况。如果目标物质为分泌物且主要在细胞外，则可选择细胞培养上清液作为样品。这里我们主要介绍细胞培养上清液样品制备的相关因素。</font></p> <p class="MsoNormal" style="text-align: center;"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;<img src="/images/upload/Image/微信图片_20250319113012.jpg" width="300" height="225" alt="elisa试剂盒" /></span></p> <h3><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">样品制备方法</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></h3> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp; &nbsp; 1、<font face="宋体">收集细胞培养上清液</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">使用</font><font face="Calibri">96</font><font face="宋体">、</font><font face="Calibri">24</font><font face="宋体">、</font><font face="Calibri">6</font><font face="宋体">孔板，（贴壁或悬浮）细、</font><font face="Calibri">6-10mm</font><font face="宋体">培养皿，</font><font face="Calibri">T25/T75</font><font face="宋体">细胞培养瓶等时，（贴壁或悬浮）细胞密度需达到</font><font face="Calibri">60-90%</font><font face="宋体">。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">悬浮细胞：在</font><font face="Calibri">2-8</font><font face="宋体">&deg;</font><font face="Calibri">C</font><font face="宋体">下以</font><font face="Calibri">2500rpm</font><font face="宋体">离心</font><font face="Calibri">5</font><font face="宋体">分钟，收集澄清的细胞培养上清液。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">贴壁细胞：直接收集上清，</font><font face="Calibri">2-8</font><font face="宋体">℃、</font><font face="Calibri">2500rpm</font><font face="宋体">离心</font><font face="Calibri">5</font><font face="宋体">分钟，收集澄清的细胞培养上清。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp; &nbsp; 2、<font face="宋体">样品预处理</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">样品通过离心、蛋白沉淀等方法进行预处理，以去除可能影响</font><font face="Calibri">ELISA</font><font face="宋体">结果的干扰物质，如细胞碎片、细胞残留物等。可通过离心将细胞碎片沉淀，并将上清液转移至新的试管中。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp; &nbsp; 3、<font face="宋体">稀释样品</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">根据</font><font face="Calibri">ELISA</font><font face="宋体">试剂盒的建议，上清液可能需要适当稀释，以确保样品浓度在试剂盒的检测范围内。浓度过高或过低都可能影响检测的灵敏度和线性范围。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp; &nbsp; 4、<font face="宋体">对照样品</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">准备阳性和阴性对照样品。阳性对照含有已知浓度的目标蛋白，用于构建标准曲线。阴性对照通常不含目标蛋白，用于检测非特异性结合和背景信号。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp; &nbsp; 5、<font face="宋体">冷冻样本</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">如果无法立即进行</font><font face="Calibri">ELISA</font><font face="宋体">检测，可以将样本冷冻保存，以确保样本的稳定性。冷冻前，请确保样本不存在因冻融引起的结构变化。尽量避免多次冻融样本，因为这可能会导致蛋白质降解和丢失。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<strong><font face="宋体">防范措施</font></strong></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">单次检测每个样本的采集体积为</font><font face="Calibri">100ul</font><font face="宋体">。如果对同一样本进行</font><font face="Calibri">N</font><font face="宋体">次检测，则需要</font><font face="Calibri">N</font><font face="宋体">个</font><font face="Calibri">100ul</font><font face="宋体">的采集体积。如果使用多个孔，则样本体积需加倍。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">样本若在一周内采集并检测，可保存于</font><font face="Calibri">2-8</font><font face="宋体">℃，若未及时检测，请分装后冷冻于</font><font face="Calibri">-20</font><font face="宋体">℃或</font><font face="Calibri">-80</font><font face="宋体">℃，避免反复冻融。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">试剂盒的检测范围不等于样品中分析物的浓度范围，建议实验前通过相关文献估算样品中分析物的浓度，并通过预实验确定样品的浓度。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">标本应新鲜检测。如果存在细菌污染，细菌可能含有内源性</font><font face="Calibri">HRP</font><font face="宋体">，也可能产生假阳性反应。如果在冰箱中保存时间过长，可能会发生聚集，导致间接</font><font face="Calibri">ELISA</font><font face="宋体">检测背景加深。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">反复冻融会降低蛋白滴度，因此若待测样本需要多次检测，应单独包装冷冻保存。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;&#10;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;&#10;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p>