小鼠孕酮(PROG)酶联免疫ELISA试剂盒
Mouse Progesterone(PROG)ELISA Kit
操作步骤：
1. 标准品的稀释与加样：在酶标包被板上设标准品孔10 孔，在*、第二孔中分别加标
准品100μl，然后在*、第二孔中加标准品稀释液50μl，混匀；然后从*孔、第二
孔中各取100μl 分别加到第三孔和第四孔，再在第三、第四孔分别加标准品稀释液50μl，
混匀；然后在第三孔和第四孔中先各取50μl 弃掉，再各取50μl 分别加到第五、第六孔
中，再在第五、第六孔中分别加标准品稀释液50ul，混匀；混匀后从第五、第六孔中各
取50μl 分别加到第七、第八孔中，再在第七、第八孔中分别加标准品稀释液50μl，混
匀后从第七、第八孔中分别取50μl 加到第九、第十孔中，再在第九第十孔分别加标准
品稀释液50μl，混匀后从第九第十孔中各取50μl 弃掉。（稀释后各孔加样量都为50μl，
浓度分别为1200pmol/L，800pmol/L ，400pmol/L，200pmol/L， 100pmol/L）。
2. 加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、待测样
品孔。在酶标包被板上待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样
品zui终稀释度为5 倍）。加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混
匀。
3. 加酶：每孔加入酶标试剂50μl，空白孔除外。
4. 温育：用封板膜封板后置37℃温育30 分钟。
5. 配液：将30（48T 的20 倍）倍浓缩洗涤液用蒸馏水30（48T 的20 倍）倍稀释后备用。
6. 洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30 秒后弃去，如此
重复5 次，拍干。
7. 显色：每孔先加入显色剂A50μl，再加入显色剂B50μl，轻轻震荡混匀，37℃避光显
色10 分钟.
8. 终止：每孔加终止液50μl，终止反应（此时蓝色立转黄色）。
9. 测定：以空白孔调零，450nm 波长依序测量各孔的吸光度（OD 值）。测定应在加终止
液后15 分钟以内进行。
Assay procedure:
1.Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, add
Standard 100μl to the first and the second well, then add Standard dilution 50μl to the first and the second well, mix; take out 100μl form the first and the second well then add it to the third and the forth well separay. then add Standard dilution 50μl to the third and the forth
well ,mix ; then take out 50μl from the third and the forth well discard, add 50μl to the fifth and the sixth well ,then add Standard dilution 50μl to the fifth and the sixth well, mix ; take out 50μl from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution 50μl to the seventh and the eighth well ,mix ; take out 50μl from the seventh and the eighth well and add to the ninth and the tenth well, add Standard dilution 50μl to the ninth and the tenth well, mix , take out 50μl from the ninth and the tenth well discard(add Sample 50μl to each well after Diluting ,(density: 1200pmol/L ， 800pmol/L ， 400pmol/L ， 200pmol/L ， 100pmol/L)
2.Add sample ： Set blank wells separay (blank comparison wells don’t add sample and
HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample
dilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is
5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.
3.Add enzyme：Add HRP-Conjugate reagent 50μl to each well, except blank well.
4.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.
5.Configurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.
6.Washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.
7.Color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the
light preservation for 10 min at 37℃
8.Stop the reaction ： Add Stop Solution50μl to each well, Stop the reaction(the blue color
change to yellow color).
9.Assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and
within 15min.
ELISA试剂盒供应商：上海华壹生物科技有限公司