酶標記物工作濃度的選擇

<p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋體;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="宋體">酶標記物包括酶標記抗原、酶標記抗體和酶標記</font>SPA<font face="宋體">等。酶標記物質量的好壞直接關系到免疫酶技術的成功與否，因此被稱為關鍵的試劑。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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;">酶標記物中常用的是酶標記抗體，它是將酶與特異性抗體經適當方法連接而成。酶標記抗體的質量主要取決于純度好、活性強及親和力高的酶和抗體，其次要有良好的制備方法。</span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">目前，高質量的酶</font>(<font face="宋體">如辣根過氧化物酶，簡稱</font><font face="Calibri">HRP)</font><font face="宋體">國內已有商品供應。高質量的抗體則可通過提取純化而獲得。在制備方法上，宜選用產率高、不影響結合物的活性和不混雜干擾性物質且操作簡便易行的方法。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;">酶聯生物</span><span style="font-family: 宋體; font-size: 9pt;">酶標記物工作濃度的選擇</span></strong></span></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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;">在免疫酶技術中，首先要確定的變異因素就是酶標記物的工作濃度。因為酶標記物濃度的很小變化，便可導致試驗結果產生很大的波動。另外，由于濃度過高，可使非特異性反應增加，而濃度過低又可影響測定的敏感性。因此，正式試驗前必須準確滴定其工作濃度。</span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">酶標記抗體的滴定方法是：將抗原</font>(<font face="宋體">或抗體</font><font face="Calibri">)</font><font face="宋體">物理吸附于固相載體上，然后將經過一系列稀釋的酶標抗體</font><font face="Calibri">(</font><font face="宋體">或抗</font><font face="Calibri">Ig</font><font face="宋體">抗體</font><font face="Calibri">)</font><font face="宋體">與吸附在載體上的抗原</font><font face="Calibri">(</font><font face="宋體">或抗體</font><font face="Calibri">)</font><font face="宋體">起反應，以酶與底物的顯色反應程度來確定酶標記抗體的效價，或稱工作濃度。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;">其步驟為：</span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">先將抗原</font>(<font face="宋體">或抗體</font><font face="Calibri">)</font><font face="宋體">用</font><font face="Calibri">0.05M PH9.6</font><font face="宋體">包被緩沖液稀釋為</font><font face="Calibri">10</font><font face="宋體">&mu;</font><font face="Calibri">g</font><font face="宋體">／</font><font face="Calibri">ml</font><font face="宋體">左右，于聚苯乙烯板孔內加</font><font face="Calibri">0.1ml</font><font face="宋體">，</font><font face="Calibri">4</font><font face="宋體">℃過夜，次日以洗滌緩沖液洗滌</font><font face="Calibri">3</font><font face="宋體">次。酶標記抗體用</font><font face="Calibri">1</font><font face="宋體">％</font><font face="Calibri">BSA-PBS</font><font face="宋體">液依次稀釋成</font><font face="Calibri">1</font><font face="宋體">∶</font><font face="Calibri">100</font><font face="宋體">，</font><font face="Calibri">1</font><font face="宋體">∶</font><font face="Calibri">200</font><font face="宋體">，</font><font face="Calibri">1</font><font face="宋體">∶</font><font face="Calibri">400</font><font face="宋體">，</font><font face="Calibri">1</font><font face="宋體">∶</font><font face="Calibri">800</font><font face="宋體">，</font><font face="Calibri">1</font><font face="宋體">∶</font><font face="Calibri">1600</font><font face="宋體">&hellip;&hellip;</font><font face="Calibri">(</font><font face="宋體">根據據抗體的滴度而定</font><font face="Calibri">)</font><font face="宋體">，分別加入反應孔中，每個稀釋度二孔，每孔</font><font face="Calibri">0.1ml</font><font face="宋體">，</font><font face="Calibri">37</font><font face="宋體">℃孵育</font><font face="Calibri">1</font><font face="宋體">小時后洗滌。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">然后加底物液，每孔</font>0.1ml<font face="宋體">，</font><font face="Calibri">37</font><font face="宋體">℃</font><font face="Calibri">10</font><font face="宋體">～</font><font face="Calibri">30</font><font face="宋體">分鐘。以</font><font face="Calibri">2M H</font><font face="宋體">２</font><font face="Calibri">SO</font><font face="宋體">４</font><font face="Calibri">0.05ml</font><font face="宋體">終止反應。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">結果判定主要以</font>ELISA<font face="宋體">比色儀讀取各孔</font><font face="Calibri">OD</font><font face="宋體">值。并以</font><font face="Calibri">OD</font><font face="宋體">為縱座標，結合物濃度為橫座標，繪制滴定曲線。由曲線上查得</font><font face="Calibri">OD</font><font face="宋體">值為</font><font face="Calibri">1.0</font><font face="宋體">左右，且曲線斜率時的酶標抗體稀釋度，即為該標記物的工作濃度。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;"><font face="宋體">有關試驗的試劑及器材均見</font>ELISA<font face="宋體">部分：</font></span></strong></span></p> <p class="MsoNormal"><span style="font-size: 9pt;"><font face="宋體" style="font-family: 宋體;">要說明的是，本法為</font><font face="宋體" style="font-family: 宋體;">ELISA直接法，所測的工作濃度與在實際應用中的</font><font face="宋體" style="font-family: 宋體;">適濃度可相差幾個滴度。這就要求在建立</font><font face="Calibri" style="font-family: 宋體;">ELISA</font><font face="宋體" style="font-family: 宋體;">實驗系統中，因此基礎上還應進一步確定實際工作濃度</font><font face="Calibri" style="font-family: 宋體;">(</font><font face="宋體" style="font-family: 宋體;">可采用方陣法</font><font face="Calibri" style="font-family: 宋體;">)</font><font face="宋體" style="font-family: 宋體;">，以達到</font><font style="" face="Calibri">合</font><font face="宋體" style="font-family: 宋體;">適的實驗條件。歡迎來電咨詢具體關于酶標記物動態。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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>