小(xiao)鼠MMP-2 ELISA試劑盒說明書(shu)

小(xiao)鼠MMP-2 ELISA試劑盒說明書(shu)

INTENDED USE AND TEST PRINCIPLE

This MMP-2 ELISA 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 of MMP-2 in the sample, this MMP-2 ELISA 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 versus MMP-2 concentration. The concentration of MMP-2 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

1.  從(cong)室溫(wen)平(ping)衡20min後的鋁箔袋(dai)中取出(chu)所(suo)需(xu)板條(tiao)，剩(sheng)余板條(tiao)用自(zi)封(feng)袋(dai)密(mi)封放回4℃。

2.  設(she)置(zhi)標(biao)準(zhun)品孔和樣本(ben)孔，標(biao)準(zhun)品孔各加(jia)不(bu)同濃(nong)度的(de)標(biao)準(zhun)品50μL；

3.  樣本(ben)孔中加(jia)入(ru)待測(ce)樣本(ben)50μL；空(kong)白(bai)孔不(bu)加(jia)。

4.  除(chu)空(kong)白孔(kong)外，標(biao)準(zhun)品孔和樣本(ben)孔中每(mei)孔加(jia)入(ru)辣根過(guo)氧化物(wu)酶（HRP）標(biao)記(ji)的檢(jian)測(ce)抗體(ti)100μL，用(yong)封(feng)板膜封住反應孔，37℃水(shui)浴(yu)鍋(guo)或恒(heng)溫(wen)箱溫(wen)育(yu)60min。

5.  棄(qi)去液體(ti)，吸(xi)水(shui)紙(zhi)上(shang)拍(pai)幹，每(mei)孔加(jia)滿(man)洗(xi)滌液（350μL），靜(jing)置(zhi)1min，甩(shuai)去洗(xi)滌液，吸(xi)水(shui)紙(zhi)上(shang)拍(pai)幹，如此重(zhong)復(fu)洗(xi)板5次（也可(ke)用洗(xi)板機洗(xi)板）。

6.  每(mei)孔加(jia)入(ru)底(di)物(wu)A、B各(ge)50μL，37℃避光(guang)孵(fu)育(yu)15min。

7.  每(mei)孔加(jia)入(ru)終止液50μL，15min內(nei)，在(zai)450nm波(bo)長(chang)處(chu)測(ce)定各孔的(de)OD值(zhi)。

實驗(yan)結(jie)果計算

以(yi)所(suo)測(ce)標(biao)準(zhun)品的OD值(zhi)為(wei)橫(heng)坐標(biao)，標(biao)準(zhun)品的濃(nong)度值(zhi)為(wei)縱(zong)坐(zuo)標(biao)，在(zai)坐標(biao)紙(zhi)上或用(yong)相(xiang)關軟(ruan)件繪制標(biao)準(zhun)曲(qu)線，並(bing)得到(dao)直線回(hui)歸方(fang)程(cheng)，將(jiang)樣品(pin)的OD值(zhi)代入方(fang)程(cheng)，計(ji)算(suan)出(chu)樣品(pin)的濃(nong)度。

試劑盒性能(neng)

1.  檢(jian)測(ce)範(fan)圍：10 ng/mL – 320 ng/mL。

2.  靈(ling)敏(min)度：檢(jian)測(ce)濃(nong)度小(xiao)於1.0 ng/mL。

3.  特(te)異(yi)性(xing)：不(bu)與(yu)其(qi)它(ta)可(ke)溶性(xing)結(jie)構類(lei)似物(wu)交叉(cha)反應。

4.  重復(fu)性(xing)：板內(nei)變(bian)異(yi)系(xi)數小(xiao)於10% ，板(ban)間(jian)變(bian)異(yi)系(xi)數小(xiao)於15% 。

ASSAY PROCEDURE

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

2.  Add 50μl of Standard or Sample to the appropriate wells. Blank well doesn’t add anyting.

3.  Add 100μl of Enzymeconjugate to standard wells and sample wells except the blank well, cover with an adhesive strip and incubate for 60 minutes at 37°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 Wash Solution (1X), then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure for a total of four times. 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 firmly when washing the plate to assure that all strips remain securely in frame.

Automated Washing - Aspirate all wells, then wash plates four times using Wash Buffer (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.  Add Substrate A 50μl and Substrate B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.

6.  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.

7.  Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.