小鼠MMP-2 ELISA试剂盒说明书

小鼠MMP-2 ELISA试剂盒说明书

INTENDEDUSEAND TEST PRINCIPLE

ThisMMP-2ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration ofMMP-2in the sample, thisMMP-2ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versusMMP-2concentration. The concentration ofMMP-2in the samples is then determined by comparing the O.D. of the samples to the standard curve.

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.样本孔中加入待测样本50μL；空白孔不加。

4.除空白孔外，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（350μL），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。

实验结果计算

以所测标准品的OD值为横坐标，标准品的浓度值为纵坐标，在坐标纸上或用相关软件绘制标准曲线，并得到直线回归方程，将样品的OD值代入方程，计算出样品的浓度。

试剂盒性能

1. 检测范围：10 ng/mL – 320 ng/mL。

2. 灵敏度：检测浓度小于1.0 ng/mL。

3. 特异性：不与其它可溶性结构类似物交叉反应。

4. 重复性：板内变异系数小于10% ，板间变异系数小于15% 。

ASSAY PROCEDURE

1.Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to theMicrotiter plate.

2.Add50μlof Standard or Sample to the appropriate wells.Blank welldoesn’t add anyting.

3.Add100μlofEnzymeconjugate to standard wellsandsample wellsexcept the blank well,cover with anadhesive stripandincubatefor60 minutesat37°C.

4.Wash the Microtiter Plate 4 times.

Manual Washing-Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well completely with WashSolution(1X), then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure for a total offourtimes. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmlywhen washing the plate to assure that all strips remain securely in frame.

Automated Washing-Aspirate all wells, then wash platesfourtimes using WashBuffer(1X). Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350μL/well/wash. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears.

5.AddSubstrateA 50μl andSubstrateB 50μl toeach well.Gently mixandincubate for 15 minutes at37°C.Protect from light.

6.Add 50μlStop Solution toeach well. The color in the wells should change from blue toyellow. If the color in the wells is green or the color change does notappear uniform,gently tap the plate to ensure thorough mixing.

7.ReadtheOpticalDensity(O.D.)at450nmusingamicrotiterplatereaderwithin15minutes.