操作注意事项
1.  试剂盒保存在2-8℃，使用前室温平衡20分钟。从冰箱取出的浓缩洗涤液会有结晶，这属于正常现象，水浴加热使结晶完全溶解后再使用。
2.  实验中不用的板条应立即放回自封袋中，密封（低温干燥）保存。
3.  预处理后的样本无需稀释，直接取10μL加样即可。
4.  严格按照说明书中标明的时间、加液量及顺序进行温育操作。
5.  所有液体组分使用前充分摇匀。

洗板方法
1.  手工洗板：甩尽孔内液体，每孔加满洗涤液，静置1min后甩尽孔内液体，在吸水纸上拍干，如此洗板5次。
2.  自动洗板机：每孔注入洗液350μL，浸泡1min，洗板5次。

操作步骤
1.  从室温平衡20min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℃。
2.  设置标准品孔和样本孔，标准品孔各加不同浓度的标准品50μL；
3.  待测样本孔先加待测样本10μL，再加样本稀释液40μL；
4.  随后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100μL，用封板膜封住反应孔，37℃水浴锅或恒温箱温育60min。
5.  弃去液体，吸水纸上拍干，每孔加满洗涤液，静置1min，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）。
6.  每孔加入底物A、B各50μL，37℃避光孵育15min。
7.  每孔加入终止液50μL，15min内，在450nm波长处测定各孔的OD值。
结果判断
 绘制标准曲线：在Excel工作表中，以标准品浓度作横坐标，对应OD值作纵坐标，绘制出标准品线性回归曲线，按曲线方程计算各样本浓度值。

免责声明
1.  试剂盒仅供研究使用，不得用于临床实验或人体实验，否则所产生的一切后果，由实验者承担，本公司概不负责。
2.  严格按照说明书操作，实验者违反说明书操作，后果由实验者承担。

Precautions
1.  Do not substitute reagents from one kit to another. Standard, conjugate ａnd microplates are matched for optimal performance. Use only the reagents supplied by manufacturer.
2.  Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.
3.  Mix all reagents before using.Remove all kit reagents from refrigerator ａnd allow them to reach room temperature ( 20-25°C)

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.

Calculation of results
1.This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis.
2.First, calculate the mean O.D. value for each standard ａnd sample. All O.D. values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software.
3.To determine the amount in each sample, first locate the O.D. value on the Y-axis ａnd extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis ａnd read the corresponding concentration.
4.Any variation in operator, pipetting ａnd washing technique, incubation time or temperature, ａnd kit age can cause variation in result. Each user should obtain their own standard curve.
5.The sensitivity by this assay is 0.1 pg/mL.
6.Standard curve.

FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!