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3. 預(yù)處理后的樣本無需稀釋，直接取 10μL 加樣即可。
4. 嚴(yán)格按照說明書中標(biāo)明的時(shí)間、加液量及順序進(jìn)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(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。
2. 自動(dòng)洗板機(jī)：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(biāo)板 | 12 孔×8 條 | 12 孔×4 條 | 無 |
| 標(biāo)準(zhǔn)品 | 0.3mL | 0.3mL | 無 |
| 樣本稀釋液 | 6mL | 3mL | 無 |
| 檢測(cè)抗體-HRP | 10mL | 5mL | 無 |
| 20×洗滌緩沖液 | 25mL | 15mL | 按說明書進(jìn)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(gè) | 1 個(gè) | 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(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. 待測(cè)樣本孔先加待測(cè)樣本 10μL，再加樣本稀釋液 40μL；
4. 隨后標(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
注：標(biāo)準(zhǔn)品濃度依次為：40、20、10、5、2.5、0 ng/mL.
的檢測(cè)抗體 100μL，用封板膜封住反應(yīng)孔，37℃水浴鍋或恒溫箱溫
試劑的準(zhǔn)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(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(zhǎng)處測(cè)定各孔的OD 值。
結(jié)果判斷
繪制標(biāo)準(zhǔn)曲線：在 Excel 工作表中，以標(biāo)準(zhǔn)品濃度作橫坐標(biāo)，對(duì)應(yīng)OD 值作縱坐標(biāo)，繪制出標(biāo)準(zhǔn)品線性回歸曲線，按曲線方程計(jì)算各樣本濃度值。
試劑盒性能
1. 準(zhǔn)確性：標(biāo)準(zhǔn)品線性回歸與預(yù)期濃度相關(guān)系數(shù) R 值，大于等于0.9900。
2. 靈敏度：檢測(cè)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%。
5. 貯藏：2-8℃，避光防潮保存。
6. 有效期：6 個(gè)月免責(zé)聲明
1. 試劑盒僅供研究使用，不得用于臨床實(shí)驗(yàn)或人體實(shí)驗(yàn)，否則所產(chǎn)生的一切后果，由實(shí)驗(yàn)者承擔(dān)，本公司概不負(fù)責(zé)。
2. 嚴(yán)格按照說明書操作，實(shí)驗(yàn)者違反說明書操作，后果由實(shí)驗(yàn)者承擔(dān)。
FOR RESEARCH USE ONLY.
NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Human TSBAb (TSBAb) ELISA Kit instruction
Intended use
This TSBAb 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 TSBAb in the sample, this TSBAb 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 TSBAb concentration. The concentration of TSBAb in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Sample collection and storages
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.
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
2. Do not remove microplate from the storage bag until needed. Unused should be stored at 2-8°C in their pouch with the desiccant provided.
3. Mix all reagents before using.
strips
Remove all kit reagents from refrigerator ａnd allow them to reach room temperature ( 20-25°C)
Materials supplied
Assay procedure
1. Prepare all reagent s before starting assay procedure. It is recommended
all Standards and Samples be added in duplicate to the Microelisa Stripplate.
that
| 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 |
2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.
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
Note: Standard concentration was followed by: 40、20、10、5、2.5、0 ng/mL.
Reagent preparation
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.
20×wash solution:Dilute with Distilled or deionized water 1:20.
6. 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.
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
FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH
ENTIRE PROCEDURE BEFORE BEGINNING!
更新時(shí)間：2024/5/24 11:45:35
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