酶标记物工作浓度的选择
<p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">酶标记物包括酶标记抗原、酶标记抗体和酶标记</font>SPA<font face="宋体">等。酶标记物质量的好坏直接关系到免疫酶技术的成功与否，因此被称为关键的试剂。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;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;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">酶标记物中常用的是酶标记抗体，它是将酶与特异性抗体经适当方法连接而成。酶标记抗体的质量主要取决于纯度好、活性强及亲和力高的酶和抗体，其次要有良好的制备方法。</span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;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;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">目前，高质量的酶</font>(<font face="宋体">如辣根过氧化物酶，简称</font><font face="Calibri">HRP)</font><font face="宋体">国内已有商品供应。高质量的抗体则可通过提取纯化而获得。在制备方法上，宜选用产率高、不影响结合物的活性和不混杂干扰性物质且操作简便易行的方法。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p>
<p class="MsoNormal"><span style="background-color: rgb(255, 255, 0);"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">酶联生物</span><span style="font-family: 宋体; font-size: 9pt;">酶标记物工作浓度的选择</span></strong></span></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;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;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">在免疫酶技术中，首先要确定的变异因素就是酶标记物的工作浓度。因为酶标记物浓度的很小变化，便可导致试验结果产生很大的波动。另外，由于浓度过高，可使非特异性反应增加，而浓度过低又可影响测定的敏感性。因此，正式试验前必须准确滴定其工作浓度。</span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;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;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">酶标记抗体的滴定方法是：将抗原</font>(<font face="宋体">或抗体</font><font face="Calibri">)</font><font face="宋体">物理吸附于固相载体上，然后将经过一系列稀释的酶标抗体</font><font face="Calibri">(</font><font face="宋体">或抗</font><font face="Calibri">Ig</font><font face="宋体">抗体</font><font face="Calibri">)</font><font face="宋体">与吸附在载体上的抗原</font><font face="Calibri">(</font><font face="宋体">或抗体</font><font face="Calibri">)</font><font face="宋体">起反应，以酶与底物的显色反应程度来确定酶标记抗体的效价，或称工作浓度。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p>
<p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">其步骤为：</span></strong></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;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;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">先将抗原</font>(<font face="宋体">或抗体</font><font face="Calibri">)</font><font face="宋体">用</font><font face="Calibri">0.05M PH9.6</font><font face="宋体">包被缓冲液稀释为</font><font face="Calibri">10</font><font face="宋体">μ</font><font face="Calibri">g</font><font face="宋体">／</font><font face="Calibri">ml</font><font face="宋体">左右，于聚苯乙烯板孔内加</font><font face="Calibri">0.1ml</font><font face="宋体">，</font><font face="Calibri">4</font><font face="宋体">℃过夜，次日以洗涤缓冲液洗涤</font><font face="Calibri">3</font><font face="宋体">次。酶标记抗体用</font><font face="Calibri">1</font><font face="宋体">％</font><font face="Calibri">BSA-PBS</font><font face="宋体">液依次稀释成</font><font face="Calibri">1</font><font face="宋体">∶</font><font face="Calibri">100</font><font face="宋体">，</font><font face="Calibri">1</font><font face="宋体">∶</font><font face="Calibri">200</font><font face="宋体">，</font><font face="Calibri">1</font><font face="宋体">∶</font><font face="Calibri">400</font><font face="宋体">，</font><font face="Calibri">1</font><font face="宋体">∶</font><font face="Calibri">800</font><font face="宋体">，</font><font face="Calibri">1</font><font face="宋体">∶</font><font face="Calibri">1600</font><font face="宋体">……</font><font face="Calibri">(</font><font face="宋体">根据据抗体的滴度而定</font><font face="Calibri">)</font><font face="宋体">，分别加入反应孔中，每个稀释度二孔，每孔</font><font face="Calibri">0.1ml</font><font face="宋体">，</font><font face="Calibri">37</font><font face="宋体">℃孵育</font><font face="Calibri">1</font><font face="宋体">小时后洗涤。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;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;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">然后加底物液，每孔</font>0.1ml<font face="宋体">，</font><font face="Calibri">37</font><font face="宋体">℃</font><font face="Calibri">10</font><font face="宋体">～</font><font face="Calibri">30</font><font face="宋体">分钟。以</font><font face="Calibri">2M H</font><font face="宋体">２</font><font face="Calibri">SO</font><font face="宋体">４</font><font face="Calibri">0.05ml</font><font face="宋体">终止反应。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;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;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">结果判定主要以</font>ELISA<font face="宋体">比色仪读取各孔</font><font face="Calibri">OD</font><font face="宋体">值。并以</font><font face="Calibri">OD</font><font face="宋体">为纵座标，结合物浓度为横座标，绘制滴定曲线。由曲线上查得</font><font face="Calibri">OD</font><font face="宋体">值为</font><font face="Calibri">1.0</font><font face="宋体">左右，且曲线斜率时的酶标抗体稀释度，即为该标记物的工作浓度。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p>
<p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;"><font face="宋体">有关试验的试剂及器材均见</font>ELISA<font face="宋体">部分：</font></span></strong></span></p>
<p class="MsoNormal"><span style="font-size: 9pt;"><font face="宋体" style="font-family: 宋体;">要说明的是，本法为</font><font face="宋体" style="font-family: 宋体;">ELISA直接法，所测的工作浓度与在实际应用中的</font><font face="宋体" style="font-family: 宋体;">适浓度可相差几个滴度。这就要求在建立</font><font face="Calibri" style="font-family: 宋体;">ELISA</font><font face="宋体" style="font-family: 宋体;">实验系统中，因此基础上还应进一步确定实际工作浓度</font><font face="Calibri" style="font-family: 宋体;">(</font><font face="宋体" style="font-family: 宋体;">可采用方阵法</font><font face="Calibri" style="font-family: 宋体;">)</font><font face="宋体" style="font-family: 宋体;">，以达到</font><font style="" face="Calibri">合</font><font face="宋体" style="font-family: 宋体;">适的实验条件。欢迎来电咨询具体关于酶标记物动态。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p>
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