小鼠促卵泡素（FSH）ELISA檢測試劑��

【資料簡介��

檢測原理

試劑盒采用雙抗體夾心法酶��(lián)免疫吸附試驗(yàn)��ELISA）。往��(yù)先包��小鼠促卵泡素��FSH）捕獲抗體的包被微孔中，依次加入��(biāo)本、標(biāo)��(zhǔn)品��HRP��(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��(zhuǎn)化成��(lán)色，并在酸的作用下轉(zhuǎn)化成最終的黃色。顏色的深淺和樣品中��小鼠促卵泡素��FSH）呈正相��(guān)。用酶標(biāo)儀��450nm 波長下測定吸光度��OD 值），計(jì)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��(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��(zhuǎn)離心30分鐘取上清��

3.  ��(xì)胞上清液��3000��(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��(zhuǎn)離心10分鐘取上清��

5.  保存：如果樣本收集后不及��(shí)檢測，請按一次用量分裝，凍存��-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(xiàng)

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

2.  ��(shí)��(yàn)中不用的板條��(yīng)立即放回自封袋中，密封（低溫干燥）保存��

3.  ��(yù)處理后的樣本無需稀釋，直接��10μL加樣即可��

4.  ��(yán)格按照說明書中標(biāo)明的��(shí)間、加液量及順序��(jìn)行溫育操作��

5.  所有液體組分使用前充分搖勻��

試劑盒組��

名稱	96孔配��	48孔配��	備注
微孔酶標(biāo)��	12��×8��	12��×4��	��
��(biāo)��(zhǔn)��	0.3mL	0.3mL	��
樣本稀釋液	6mL	3mL	��
檢測抗體-HRP	10mL	5mL	��
20×洗滌緩沖��	25mL	15mL	按說明書��(jìn)行稀��
底物A	6mL	3mL	��
底物B	6mL	3mL	��
終止��	6mL	3mL	��
封板��	2��	2��	��
說明��	1��	1��	��
自封��	1��(gè)	1��(gè)	��

注：��(biāo)��(zhǔn)品濃度依次為��80��40��20��10��5��0 mIU/mL.

試劑的準(zhǔn)��

 20×洗滌緩沖液的稀釋：蒸餾水按1��20稀釋，��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��(nèi)液體，每孔加滿洗滌液，靜��1min后甩盡孔��(nèi)液體，在吸水紙上拍干，如此洗��5次��

2.  自動洗板��(jī)：每孔注入洗��350μL，浸��1min，洗��5次��

操作步驟

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

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

3.  待測樣本孔先加待測樣��10μL，再加樣本稀釋液40μL��

4.  隨后��(biāo)��(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��(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(jī)洗板）��

6.  每孔加入底物A、B��50μL��37℃避光孵��15min��

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

��(jié)果判��

 繪制��(biāo)��(zhǔn)曲線：在Excel工作表中，以��(biāo)��(zhǔn)品濃度作橫坐��(biāo)，對��(yīng)OD值作縱坐��(biāo)，繪制出��(biāo)��(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.  ��(zhǔn)確性：��(biāo)��(zhǔn)品線性回歸與��(yù)期濃度相��(guān)系數(shù)R值，大于等于0.9900��

2.  靈敏度：最di檢測濃度小于0.1 mIU/mL��

3.  檢測范圍��2.5 mIU/mL - 80 mIU/mL                                            

4. 特異性：不與其它可溶性結(jié)��(gòu)類似物交叉反��(yīng)��

5.  重復(fù)性：板內(nèi)變異系數(shù)小于10%、板間變異系��(shù)小于15%��

6.  貯藏��2-8℃，避光防潮保存��

7.  有效期：6��(gè)��

免責(zé)聲明

1.   試劑盒僅供研究使用，不得用于臨床��(shí)��(yàn)或人體實(shí)��(yàn)，否則所��(chǎn)生的一切后果，由實(shí)��(yàn)者承��(dān)，本公司概不��(fù)��(zé)��

2.   ��(yán)格按照說明書操作，實(shí)��(yàn)者違反說明書操作，后果由��(shí)��(yàn)者承��(dān)��

FOR RESEARCH USE ONLY. 

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

 

Mouse follicle-stimulating hormone (FSH) ELISA Kit instruction

 

Intended use

This FSH 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 FSH in the sample, this FSH 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 FSH concentration. The concentration of FSH 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 should be centrifugated adequately 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	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

Note: Standard concentration was followed by:

80��40��20��10��5��0 mIU/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. 

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 towels.

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 mIU/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!