小鼠（Mouse）免疫球蛋白 G（IgG）ELISA 檢測試劑盒 使用說明書

【資料簡介】

本試劑盒只能用于科學(xué)研究，不得用于醫(yī)學(xué)診斷
小鼠（Mouse）免疫球蛋白 G（IgG）ELISA 檢測試劑盒
小鼠（Mouse）免疫球蛋白 G（IgG）ELISA 檢測試劑盒
使用說明書
檢測原理
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)
先包被免疫球蛋白G（IgG）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*洗滌。 用底物TMB顯色，
TMB在過氧化物酶的催化下轉(zhuǎn)化成藍色， 并在酸的作用下轉(zhuǎn)化成zui終
的黃色。顏色的深淺和樣品中的免疫球蛋白G（IgG）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。
樣品收集、處理及保存方法
1. 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(xì)胞
刺激，收集血液后，3000 轉(zhuǎn)離心 10 分鐘將血清和紅細(xì)胞迅速小心地
分離。
2. 血漿： EDTA、 檸檬酸鹽或肝素抗凝。 3000 轉(zhuǎn)離心 30 分鐘取上清。
3. 細(xì)胞上清液：3000 轉(zhuǎn)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(zhuǎn)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(fù)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(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
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(jié)晶，這屬于正常現(xiàn)象，水浴加熱使結(jié)晶*溶解
后再使用。
2. 實驗中不用的板條應(yīng)立即放回自封袋中， 密封 （低溫干燥） 保存。
3. 濃度為 0 的 S0 號標(biāo)準(zhǔn)品即可視為陰性對照或者空白；按照說明
書操作時樣本已經(jīng)稀釋 5 倍，zui終結(jié)果乘以 5 才是樣本實際濃度。
4. 嚴(yán)格按照說明書中標(biāo)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
試劑盒組成
名稱  96 孔配置  48 孔配置 備注
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*6 管  0.3mL*6 管 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
注：標(biāo)準(zhǔn)品（S0-S5）濃度依次為：0、1.25、2.5、5、10、20 mg/mL
試劑的準(zhǔn)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
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. 自動洗板機：每孔注入洗液 350μL，浸泡 1min，洗板 5 次。
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. 樣本孔先加待測樣本 10μL，再加* 40μL；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，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。
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：zui低檢測濃度小于 0.1 mg/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)、板間變異系數(shù)均小于 15%。
5. 貯藏：2-8℃，避光防潮保存。
6. 有效期：6 個月
免責(zé)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(chǎn)生的一切后果，由實驗者承擔(dān)，本公司概不負(fù)責(zé)。
2. 嚴(yán)格按照說明書操作，實驗者違反說明書操作，后果由實驗者
承擔(dān)。
FOR RESEARCH USE ONLY.
NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Mouse Immunoglobulin G (IgG) ELISA Kit
instruction
Intended use
This IgG 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 IgG in the sample, this
IgG 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 IgG concentration. The concentration of
IgG 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 approximay 3000×g. Remove serum and
assay immediay 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 immediay or aliquot and store samples at -20 or  ℃
-80 . Avoid repeated freeze ℃ -thaw cycles.
Note: The samples shoule be centrifugated dequay 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
( 20-25°C)
Materials supplied
Name  96 determinations 48 determinations
Microelisa stripplate  12*8strips  12*4strips
Standard  0.3ml*6tubes  0.3ml*6tubes
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
Note:  Standard (S0 → S5) concentration was followed by:0,1.25,2.5,5,10,20
mg/ml
Reagent preparation
20×wash solution:Dilute with Distilled or deionized water 1:20.
Assay procedure
1. Prepare all reagents before starting assay procedure. It is recommended that
all Standards and Samples be added in duplicate to the Microelisa Stripplate.
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.
4. 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.
5. 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.
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 (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 mg/ml
6.  Standard curve
Storage： 2-8℃.
validity： six months.
FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR
DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH
ENTIRE PROCEDURE BEFORE BEGINNING!