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日本MBL公司人嗜酸细胞阳离子蛋白(ECP)试剂盒说明书 

For Research Use Only. Not for use in diagnostic procedures.

2. Summary and Explanation Eosinophil Cationic protein (ECP), Eosinophil derived Neurotoxin (EDN) and Major Basic Protein (MBP) are known to be major protein-mediators derived from activated eosinophils. ECP and EDN are found in matrix of granules in eosinophils whereas MBP is found in core of granules. ECP and EDN are members of the ribonuclease A superfamily. ECP and MBP have high cytotoxicity. These three proteins are highly cationic proteins with PI 10.8-10.9. Activated eosinophile play an important role in the late asthmatic response and in the asthmatic airway inflammation. As ECP is secreted from activated eosinophils, ECP can be a marker of eosinophile activation and degranulation.“MESACUP ECP TEST” is the reagent for measuring specifically with high sensitivity ECP by ELISA.

3. Principle

MBL MESACUP ECP TEST measures human ECP by sandwich ELISA. This ELISA detects human ECP with a minimum detection limit of 0.125 ng/ml and does not cross-react with EDN. In the wells coated with anti-human ECP monoclonal antibody, samples to be measured or standards are incubated. After washing, a peroxidase conjugated anti-human ECP polyclonal antibody is added into the microwells and incubated. After another washing, the peroxidase substrate is mixed with the chromogen and allowed to incubate for an additional period of time. An acid solution is then added to each well to terminate the enzyme reaction and to stabilize the developed color. The optical density (O.D.) of each well is then measured at 450 nm using a microplate reader. The concentration of ECP is calibrated from a standard curve based on reference standards.

人基质金属蛋白酶9/明胶酶BELISA试剂盒操纵步骤：

1. 标准品的稀释与加样：在酶标包被板上设标准品孔10孔，在第一、第二孔中分别加标准品100μl，然后在第一、第二孔中加标准品稀释液50μl，混匀；然后从第一孔、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3000ng/L，2000ng/L ，1000ng/L，500ng/L， 250ng/L）。

2. 加样：分别设空缺孔（空缺对照孔不加样品及酶标试剂，其余各步操纵相同）、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（样品终极稀释度为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. 温育：用封板膜封板后置37℃温育30分钟。

4. 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。

5. 洗涤：小心揭掉封板膜，弃往液体，甩干，每孔加满洗涤液，静置30秒后弃往，如此重复5次，拍干。

6. 加酶：每孔加进酶标试剂50μl，空缺孔除外。

7. 温育：操纵同3。

8. 洗涤：操纵同5。

9. 显色：每孔先加进显色剂A50μl，再加进显色剂B50μl，轻轻震荡混匀，37℃避光显色15分钟.

10. 终止：每孔加终止液50μl，终止反应（此时蓝色立转黄色）。

11. 测定：以空缺空调零，450nm波长依序丈量各孔的吸光度（OD值）。 测定应在加终止液后15分钟以内进行。
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