本試劑盒只能用于科學研究，不得用于醫(yī)學診斷
犬抗核抗體（ANA）ELISA 檢測試劑盒
使用說明書
檢測原理
試劑盒采用間接法酶聯(lián)免疫吸附試驗（ELISA）。往預先包被犬抗核抗體（ANA）捕獲抗原的包被微孔中，依次加入標本、標準品、
HRP標記的檢測抗體，經(jīng)過溫育并*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犬抗核抗體（ANA）呈正相關。用酶標儀在450nm 波長下測定吸光度（OD 值），判斷樣品是否含有犬抗核抗體（ANA）。
樣品收集、處理及保存方法
- 血清：使用不含熱原和內(nèi)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。
- 血漿：EDTA、檸檬酸鹽或肝素抗凝。3000 轉離心 30 分鐘取上清。
- 細胞上清液：3000 轉離心 10 分鐘去除顆粒和聚合物。
- 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
- 高精度加樣器及槍頭：0.5-10uL、2-20uL、20-200uL、200-1000uL
操作注意事項
- 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*溶解后再使用。
- 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。
- 預處理后的樣本無需稀釋，直接取 10μL 加樣即可。
- 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。
試劑盒組成
名稱 | 96 孔配置 | 48 孔配置 | 備注 |
| | | |
微孔酶標板 | 96 孔 | 48 孔 | 無 |
| | | |
陰性對照 | 0.3mL | 0.3mL | 無 |
| | | |
陽性對照 | 0.3mL | 0.3mL | 無 |
| | | |
* | 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 |
| | | |
試劑的準備
20×洗滌緩沖液的稀釋：蒸餾水按 1：20 稀釋，即 1 份的 20×洗滌
緩沖液加 19 份的蒸餾水。
洗板方法
- 手工洗板：甩盡孔內(nèi)液體，每孔加滿洗滌液，靜置 1min 后甩盡孔內(nèi)液體，在吸水紙上拍干，如此洗板 5 次。
- 自動洗板機：每孔注入洗液 350μL，浸泡 1min，洗板 5 次。
操作步驟
- 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℃。
- 設置陰、陽性對照孔和樣本孔，陰、陽性對照孔中加入陰性對照、陽性對照各 50μL；
- 待測樣本孔先加待測樣本 10μL，再加* 40μL；
- 隨后陰、陽性對照孔和樣本孔中每孔加入辣根過氧化物酶（HRP）標記的檢測抗原 100μL，用封板膜封住反應孔，37℃水浴鍋或恒溫箱溫育 60min。
- 棄去液體，吸水紙上拍干，每孔加滿洗滌液，靜置 1min，甩去洗滌液，吸水紙上拍干，如此重復洗板 5 次（也可用洗板機洗板）。
- 每孔加入底物 A、B 各 50μL，37℃避光孵育 15min。
- 每孔加入終止液 50μL，15min 內(nèi)，在 450nm 波長處測定各孔的
OD 值。
結果判斷
- 試驗有效性：陽性對照孔 OD 值平均值≥1.00；
陰性對照孔 OD 值平均值≤0.15。
- 臨界值（Cut off）計算：臨界值=陰性對照孔平均值+0.15
- 陰性判斷：樣品 OD 值＜臨界值（Cut off），樣品為陰性
- 陽性判斷：樣品 OD 值＞臨界值（Cut off），樣品為陽性
試劑盒性能
- 準確性：陽性對照孔 OD 值平均值≥1.00；陰性對照孔 OD 值平均值≤0.15，說明試驗結果有效。
- 重復性：板內(nèi)、板間變異系數(shù)均小于 15%。
免責聲明
- 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(chǎn)生的一切后果，由實驗者承擔，本公司概不負責。
- 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.
Canine Anti-nuclear Antibody (ANA) ELISA Kit
instruction
Intended use
This ANA 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 ANA in the sample, this ANA 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 ANA concentration. The concentration of ANA 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
- Standard microplate reader(450nm)
- Precision pipettes and Disposable pipette tips.
Precautions
- 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.
- 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.
- Mix all reagents before using.
Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C)
Materials supplied
Name | 96 determinations | 48 determinations |
| | |
Microelisa stripplate | 96 strips | 48 strips |
Negative control | 0.3ml | 0.3ml |
| | |
Positive control | 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 |
| | |
Reagent preparation
20×wash solution:Dilute with Distilled or deionized water 1:20.
Assay procedure
1. Prepare all r e a g e n t s before starting assay procedure. It is recommended that
all Standards and Samples be added in duplicate to the Microelisa Stripplate.
- Separately add Positive control and Negative control 50μl to the Positive and Negative well, Add testing sample 10μl Then add sample diluent 40μl to testing sample well; Blank well doesn’t add anyting.
- Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C.
- 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.
- 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.
- 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.
- Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
