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Human DMD ELISA Kit

For the quantitative in vitro determination of Human Dystrophin concentrations in

 serum - plasma - celiac fluid - tissue homogenate - body fluid

FOR LABORATORY RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES. 

This package ｉnsert must be read in its entirety before using this product.

ELISA

ENZYME LINKED IMMUNOSORBENT ASSAY

INTENDED USE AND TEST PRINCIPLE

This DMD 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 DMD in the sample, this DMD 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 DMD concentration. The concentration of DMD in the samples is then determined by comparing the O.D. of the samples to the standard curve.

SAMPLE COLLECTION AND STORAGES

Serum - Use a serum separator tube ａnd allow samples to clot for 2 hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximately 2000×g. Remove serum ａnd assay immediately or aliquot ａnd store samples at -20℃. Avoid repeated freeze-thaw cycles

Plasma - Collect plasma using heparin as an anticoagulant. Centrifuge samples for 30 minutes at 2000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃. Avoid repeated freeze-thaw cycles.

Cell culture supernates, tissue homogenate and other biological fluids - Remove particulates by centrifugation ａnd assay immediately or aliquot ａnd store samples at -20℃. Avoid repeated freeze-thaw cycles.

Note:  The samples shoule be centrifugated dequately ａnd no hemolysis or granule was allowed.

MATERIALS REQUIRED BUT NOT SUPPLIED

1.  37 ℃ incubator

2.  Standard microplate reader capable of measuring absorbance at 450 nm

3.  Precision pipettes, disposable pipette tips and Absorbent paper

4.  Distilled or deionized water

REAGENTS PROVIDED 

All reagents provided are stored at 2-8°C. Refer to the expiration date on the label.

Note:

1.  Standard concentration was followed by: 8, 4, 2, 1, 0.5, 0 ng/mL.

2.  If samples generate values higher than the highest standard, please dilute the samples with Sample Diluent and repeat the assay.

PRECAUTIONS

1. Do not substitute reagents from one kit lot to another. Standard, conjugate ａnd microtiter plates are matched for optimal performance. Use only the reagents supplied by manufacturer.

2. Allow kit reagents ａnd materials to reach room temperature (20-25°C) before use. Do not use water baths to thaw samples or reagents.

3. Do not use kit components beyond their expiration date.

4. Use only deionized or distilled water to dilute reagents.

5. Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.

6. Use fresh disposable pipette tips for each transfer to avoid contamination.

7. Do not mix acid ａnd sodium hypochlorite solutions.

8. Serum ａnd plasma should be handled as potentially hazardous ａnd capable of transmitting disease. Disposable gloves must be worn during the assay procedure, since no known test method can offer complete assurance that products derived from Rat blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious ａnd good laboratory practices should be followed.

9. All samples should be disposed of in a manner that will inactivate viruses.

10. Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal.

11. Substrate Solution is easily contaminated. If bluish prior to use, do not use.

12. Substrate B contain 20% acetone, keep this reagent away from sources of heat or flame.

13. Remove all kit reagents from refrigerator ａnd allow them to reach room temperature ( 20-25°C).

REAGENT PREPARATION AND STORAGE

Wash Solution (1X) - Dilute 1 volume of Wash solution (20X) with 19 volumes of deionized or distilled water. Wash Solution is stable for 1 month at 2-8°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 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.

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 (X) axis versus the corresponding concentration on the horizontal (Y) 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 balnk well 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. Intra-assay CV(%) is less than 10% ａnd Inter-assay CV(%) is less than 15%.

6. Assay range: 0.25 ng/mL – 8 ng/mL.

7.  Sensitivity: The minimum detectable dose of Human DMD is typically less than 0.1 ng/mL.

8.  Cross-reactivity: This assay recognizes recombinant ａnd natural Human DMD. No significant cross-reactivity or interference was observed.

9.  Storage: 2-8℃ (Use frequently); six months (-20℃)。

10.  Standard curve

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

人肌營養(yǎng)不良蛋白（DMD）試劑盒（ELISA）

使用說明書

l 本試劑盒用于體外定量檢測血清、血漿、組織、細(xì)胞上清及相關(guān)液體樣本中人肌營養(yǎng)不良蛋白（DMD）的含量。

l 有效期：6個月

l 保存條件：2-8℃

實驗原理

試劑盒采用雙抗體一步夾心法酶聯(lián)免疫吸附試驗（ELISA）。往預(yù)先包被人肌營養(yǎng)不良蛋白（DMD）捕獲抗體的包被微孔中，依次加入標(biāo)本、標(biāo)準(zhǔn)品、HRP標(biāo)記的檢測抗體，經(jīng)過溫育并徹底洗滌。用底物TMB顯色，TMB在過氧化物酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸的作用下轉(zhuǎn)化成*終的黃色。顏色的深淺和樣品中的人肌營養(yǎng)不良蛋白（DMD）呈正相關(guān)。用酶標(biāo)儀在450nm 波長下測定吸光度（OD 值），計算樣品濃度。

樣本處理及要求

1.  血清：全血標(biāo)本請于室溫放置2小時或4℃過夜后于1000g離心20分鐘，取上清即可檢測，或?qū)��?biāo)本放于-20℃或-80℃保存，但應(yīng)避免反復(fù)凍融。
2.  血漿：可用EDTA或肝素作為抗凝劑，標(biāo)本采集后30分鐘內(nèi)于2 - 8°C 1000g離心20分鐘，或?qū)��?biāo)本放于-20℃或-80℃保存，但應(yīng)避免反復(fù)凍融。
3.  細(xì)胞培養(yǎng)物上清或其它生物標(biāo)本：1000g離心20分鐘，取上清即可檢測，或?qū)��?biāo)本放于-20℃或-80℃保存，但應(yīng)避免反復(fù)凍融。
注：標(biāo)本溶血會影響*后檢測結(jié)果，因此溶血標(biāo)本不宜進(jìn)行此項檢測。

需要而未提供的試劑和器材

1. 酶標(biāo)儀（450nm）

2. 高精度加樣器及槍頭：0.5-10uL、2-20uL、20-200uL、200-1000uL

3. 37℃恒溫箱

4. 蒸餾水或去離子水

試劑盒組成

備注：

1.  標(biāo)準(zhǔn)品濃度依次為：8、4、2、1、0.5、0 ng/mL

2.  經(jīng)過大量正常標(biāo)本檢驗，標(biāo)本的正常濃度值均在試劑盒提供的檢測范圍內(nèi)，實驗過程中直接取50μL樣本上樣即可。當(dāng)有部分樣本值超過標(biāo)準(zhǔn)品濃度時，可用樣本稀釋液將標(biāo)本進(jìn)行適當(dāng)稀釋后再進(jìn)行實驗。

注意事項

1. 嚴(yán)格按照規(guī)定的時間和溫度進(jìn)行溫育以保證準(zhǔn)確結(jié)果。所有試劑都必須在使用前達(dá)到室溫20-25℃。使用后立即冷藏保存試劑。

2. 洗板不正確可以導(dǎo)致不準(zhǔn)確的結(jié)果。在加入底物前確保盡量吸干孔內(nèi)液體。溫育過程中不要讓微孔干燥掉。

3. 消除板底殘留的液體和手指印，否則影響OD值。

4. 底物顯色液應(yīng)呈無色或很淺的顏色，已經(jīng)變藍(lán)的底物液不能使用。

5. 避免試劑和標(biāo)本的交叉污染以免造成錯誤結(jié)果。

6. 在儲存和溫育時避免強(qiáng)光直接照射。

7. 平衡至室溫后再打開密封袋以防水滴凝聚在冷板條上。

8. 任何反應(yīng)試劑不能接觸漂白溶劑或漂白溶劑所散發(fā)的強(qiáng)烈氣體。任何漂白成分都會破壞試劑盒中反應(yīng)試劑的生物活性。

9. 不能使用過期產(chǎn)品。

10. 如果可能傳播疾病，所有的樣品都應(yīng)管理好，按照規(guī)定的程序處理樣品和檢測裝置。

試劑準(zhǔn)備

試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡后方可使用。

20×洗滌緩沖液的稀釋：蒸餾水按1：20稀釋，即1份20×洗滌緩沖液加19份蒸餾水。

操作步驟

1.  從室溫平衡20min后的鋁箔袋中取出所需板條，剩余板條用自封袋密封放回4℃。

2.  設(shè)置標(biāo)準(zhǔn)品孔和樣本孔，標(biāo)準(zhǔn)品孔各加不同濃度的標(biāo)準(zhǔn)品50μL；

3.  樣本孔中加入待測樣本50μL；空白孔不加。

4.  除空白孔外，標(biāo)準(zhǔn)品孔和樣本孔中每孔加入辣根過氧化物酶（HRP）標(biāo)記的檢測抗體100μL，用封板膜封住反應(yīng)孔，37℃水浴鍋或恒溫箱溫育60min。

5.  棄去液體，吸水紙上拍干，每孔加滿洗滌液（350μL），靜置1min，甩去洗滌液，吸水紙上拍干，如此重復(fù)洗板5次（也可用洗板機(jī)洗板）。

6.  每孔加入底物A、B各50μL，37℃避光孵育15min。

7.  每孔加入終止液50μL，15min內(nèi)，在450nm波長處測定各孔的OD值。

實驗結(jié)果計算

以所測標(biāo)準(zhǔn)品的OD值為橫坐標(biāo)，標(biāo)準(zhǔn)品的濃度值為縱坐標(biāo)，在坐標(biāo)紙上或用相關(guān)軟件繪制標(biāo)準(zhǔn)曲線，并得到直線回歸方程，將樣品的OD值代入方程，計算出樣品的濃度。

試劑盒性能

1.  檢測范圍：0.25 ng/mL – 8 ng/mL。

2.  靈敏度：檢測濃度小于0.1 ng/mL。

3.  特異性：不與其它可溶性結(jié)構(gòu)類似物交叉反應(yīng)。

4.  重復(fù)性：板內(nèi)變異系數(shù)小于10% ，板間變異系數(shù)小于15% 。