ELISA 檢測試劑盒#人解整合素樣金屬蛋白酶 12（ADAM12）ELISA 檢測試劑盒2022年產品說明書#技術新聞

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ELISA 檢測試劑盒#人解整合素樣金屬蛋白酶 12（ADAM12）ELISA 檢測試劑盒2022年產品說明書#技術新聞
據技術部4月25日14時15分透露：（http://www.app17.com/c110001/products/d11333577.html）

檢測原理

試劑盒采用雙抗體夾心法酶聯(lián)免疫吸附試驗（ELISA）。往預先包被人解整合素樣金屬蛋白酶12（ADAM12）捕獲抗體的包被微孔中，依次加入標本、標準品、HRP標記的檢測抗體，經過溫育并徹*底洗滌。用底物TMB顯色，TMB在過氧化物酶的催化下轉化成藍色，并在酸的作用下轉化成*終的黃色。顏色的深淺和樣品中的人解整合素樣金屬蛋白酶12（ADAM12）呈正相關。用酶標儀在450nm 波長下測定吸光度（OD 值），計算樣品濃度。

樣品收集、處理及保存方法

1. 血清：使用不含熱原和內毒素的試管，操作過程中避免任何細胞刺激，收集血液后，3000 轉離心 10 分鐘將血清和紅細胞迅速小心地分離。

2. 血漿：EDTA、檸檬酸鹽或肝素抗凝。3000 轉離心 30 分鐘取上清。

3. 細胞上清液：3000 轉離心 10 分鐘去除顆粒和聚合物。

4. 組織勻漿：將組織加入適量生理鹽水搗碎。3000 轉離心 10 分鐘取上清。

5. 保存：如果樣本收集后不及時檢測，請按一次用量分裝，凍存于

-20℃，避免反復凍融，在室溫下解凍并確保樣品均勻地充分解凍。自備物品

1. 酶標儀（450nm）

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

3. 37℃恒溫箱操作注意事項

1. 試劑盒保存在 2-8℃，使用前室溫平衡 20 分鐘。從冰箱取出的濃縮洗滌液會有結晶，這屬于正�，F(xiàn)象，水浴加熱使結晶完*全溶解后再使用。

2. 實驗中不用的板條應立即放回自封袋中，密封（低溫干燥）保存。

3. 預處理后的樣本無需稀釋，直接取 10μL 加樣即可。

4. 嚴格按照說明書中標明的時間、加液量及順序進行溫育操作。

5. 所有液體組分使用前充分搖勻。

試劑盒組成

洗板方法

1. 手工洗板：甩盡孔內液體，每孔加滿洗滌液，靜置 1min 后甩盡孔內液體，在吸水紙上拍干，如此洗板 5 次。

2. 自動洗板機：每孔注入洗液 350μL，浸泡 1min，洗板 5 次。

名稱	96 孔配置	48 孔配置	備注
微孔酶標板	12 孔×8 條	12 孔×4 條	無
標準品	0.3mL	0.3mL	無
樣本稀釋液	6mL	3mL	無
檢測抗體-HRP	10mL	5mL	無
20×洗滌緩沖液	25mL	15mL	按說明書進行稀釋
底物 A	6mL	3mL	無
底物 B	6mL	3mL	無
終止液	6mL	3mL	無
封板膜	2 張	2 張	無
說明書	1 份	1 份	無
自封袋	1 個	1 個	無

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. 設置標準品孔和樣本孔，標準品孔各加不同濃度的標準品 50μL；

3. 待測樣本孔先加待測樣本 10μL，再加樣本稀釋液 40μL；

4. 隨后標準品孔和樣本孔中每孔加入辣根過氧化物酶（HRP）標記

注：標準品濃度依次為：16、8、4、2、1、0 ng/mL.

的檢測抗體 100μL，用封板膜封住反應孔，37℃水浴鍋或恒溫箱溫

試劑的準備

育 60min。

20×洗滌緩沖液的稀釋：蒸餾水按 1：20 稀釋，即 1 份的 20×洗滌

5. 棄去液體，吸水紙上拍干，每孔加滿洗滌液，靜置 1min，甩去

緩沖液加 19 份的蒸餾水。

洗滌液，吸水紙上拍干，如此重復洗板 5 次（也可用洗板機洗板）。

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，在 450nm 波長處測定各孔的OD 值。

結果判斷

繪制標準曲線：在 Excel 工作表中，以標準品濃度作橫坐標，對應OD 值作縱坐標，繪制出標準品線性回歸曲線，按曲線方程計算各樣本濃度值。

試劑盒性能

1. 準確性：標準品線性回歸與預期濃度相關系數 R 值，大于等于0.9900。

2. 靈敏度：**低檢測濃度小于 0.1 ng/mL。

3. 特異性：不與其它可溶性結構類似物交叉反應。

4. 重復性：板內變異系數小于 10%、板間變異系數小于 15%。

5. 貯藏：2-8℃，避光防潮保存。

6. 有效期：6 個月免責聲明

1. 試劑盒僅供研究使用，不得用于臨床實驗或人體實驗，否則所產生的一切后果，由實驗者承擔，本公司概不負責。

2. 嚴格按照說明書操作，實驗者違反說明書操作，后果由實驗者承擔。

FOR RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Human A Disintegrin And Metalloprotease

12 (ADAM12) ELISA Kit instruction

Intended use

This ADAM12 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 ADAM12 in the sample, this ADAM12 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 ADAM12 concentration. The concentration of ADAM12 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 30 minutes

before centrifugation for 10 minutes at approximately 3000×g. Remove serum and

assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Cell culture supernates and other biological fluids - Remove particulates

by centrifugation and assay immediately or aliquot and store samples at -20℃or

-80℃. 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. Standard microplate reader(450nm)

2. Precision pipettes and Disposable pipette tips.

3. 37 ℃ incubator

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

Assay procedure

1. Prepare all reagent s before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.

( 20-25°C)

Materials supplied

2. Add standard: Set Standard wells, testing sample wells.

standard well.

Add standard 50μl to

Name	96 determinations	48 determinations
Microelisa stripplate	12*8strips	12*4strips
Standard	0.3ml	0.3ml
Sample diluent	6.0ml	3.0ml
HRP-Conjugate reagent	10.0ml	5.0ml
20X Wash solution	25ml	15ml
Chromogen Solution A	6.0ml	3.0ml
Chromogen Solution B	6.0ml	3.0ml
Stop Solution	6.0ml	3.0ml
Closure plate membrane	2	2
User manual	1	1
Sealed bags	1	1

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

Note: Standard concentration was followed by:towels.

16、8、4、2、1、0 ng/mL.

6. Add chromogen solution A 50μl and chromogen solution B 50μl to each well.

Reagent preparation

Gently mix ａnd incubate for 15 minutes at 37°C. Protect from light.

20×wash solution:Dilute with Distilled or deionized water 1:20.

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 ng/mL.

6. Standard curve