上海轩泽康生物有限公司猪透明质酸(HA)ELISA试剂盒操作步骤如下：
步骤一： | 从室温平衡60分钟后的铝箔袋中取出所需板条，剩余板条用自封袋密封放回4℃。 |
步骤二： | 标准品的加样：设置标准品孔和样本孔，标准品孔各加不同浓度的标准品50ul。 |
步骤三： | 样本孔中加入待测样本50ul；空白孔不加。 |
步骤四： | 每孔加入生物素标记抗体50ul，用封板膜封住反应孔，37℃水浴锅或恒温箱温育30分钟；标准孔空白孔不加。 |
步骤五： | 弃去液体，吸水纸上拍干，每孔加满洗涤液（350ul），静置1分钟，甩去洗涤液，吸水纸上拍干，如此重复洗板5次（也可用洗板机洗板）。 |
步骤六： | 除去空白孔外，标准品孔和样本孔中每孔加入辣根过氧化物酶（HRP）标记的检测抗体100ul，用封板膜封住反应孔，37℃水浴锅或恒温箱温育30分钟。 |
步骤七： | 重复步骤五 |
步骤八： | 每孔加入底物A、B各50ul，37℃避光孵育15分钟。 |
步骤九： | 每孔加入终止液50ul，15分钟内在450nm波长处测定各孔的OD值。 |
Assay procedure
1. Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.
2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.
3. Add Sample: Add testing sample 10μl then add Sample Diluent 40μl to testing sample well; Blank well doesn’t add anyting.
4. Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C.
5. Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.
7. Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not
appear uniform, gently tap the plate to ensure thorough mixing.
8. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.