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廈門(mén)慧嘉生物科技有限公司
初級(jí)會(huì)員 | 第10年

18906011628

(中英文)大鼠促卵泡素(FSH)ELISA Kit使用操作說(shuō)明書(shū)

時(shí)間:2013/5/12閱讀:382
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Rat Follicle-Stimulating
Hormone(FSH)Elisa Kit  
 
 
 
 
Catalog No. CSB-E06869r
(48T)
 
 
 
 
 
  This immunoassay kit allows for the in vitro quantitative determination of rat FSH
concentrations in serum, plasma and other biological fluids.
  Expiration date      six months from the date of manufacture
  FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
 
 
 
  2
INTRODUCTION
Follicle-stimulating hormone (FSH) is a hormone synthesized and secreted
by  gonadotropes  in  the  anterior  pituitary  gland.  FSH  regulates  the
development, growth, pubertal maturation, and  reproductive processes of
the human body. FSH and Luteinizing hormone  (LH) act synergistically  in
reproduction.
FSH  is a glycoprotein. Each monomeric unit  is a protein molecule with a
sugar  attached  to  it;  two  of  these  make  the  full,  functional  protein.  Its
structure  is  similar  to  those  of  LH,  TSH,  and  hCG.  The  protein  dimer
contains 2 polypeptide units,  labeled alpha and beta  subunits. The alpha
subunits of LH, FSH, TSH, and hCG are  identical, and contain 92 amino
acids. The beta subunits vary. FSH has a beta subunit of 118 amino acids
(FSHB),  which  confers  its  specific  biologic  action  and  is  responsible  for
interaction  with  the  FSH-receptor.  The  sugar  part  of  the  hormone  is
composed  of  fucose,  galactose,  mannose,  galactosamine,  glucosamine,
and sialic acid, the latter being critical for its biologic half-life. The half-life of
FSH is 3-4 hours. Its molecular wt is 30000.
PRINCIPLE OF THE ASSAY
The  microtiter  plate  provided  in  this  kit  has  been  pre-coated  with  a
goat-anti-rabbit  antibody.  Standards  or  samples  are  then  added  to  the
appropriate  microtiter  plate  wells  with  a  Horseradish  Peroxidase
(HRP)-conjugated  FSH  and  anti-FSH  antibody  and  incubated.  Then
substrate solution A and B are added  to each well. The enzyme-substrate
reaction  is  terminated by  the addition of a sulphuric acid solution and  the
color  change  is measured  spectrophotometrically  at  a wavelength  of  450
nm ± 2 nm. The concentration of FSH in the samples is then determined by
comparing the O.D. of the samples to the standard curve. 
  3
DETECTION RANGE
0.5 mIU/ml-100 mIU/ml.  The  standard  curve  concentrations  used  for  the
ELISA’s were 100 mIU/ml, 40 mIU/ml, 10 mIU/ml, 2 mIU/ml, 0.5 mIU/ml.
SPECIFICITY
This  assay  recognizes  recombinant  and  natural  rat  FSH.  No  significant
cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of rat FSH is typically less than 0.2 mIU/ml.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined
as the lowest concentration that could be differentiated from zero.
MATERIALS PROVIDED
Reagent      Quantity
Assay plate  1
Standard(S1-S5)  5
Antibody  1 x 3 ml
HRP-conjugate  1 x 3 ml
Wash Buffer      
1 x 15 ml
  (20×concentrate)
Substrate A  1 x 3.5 ml
Substrate B  1 x 3.5 ml
Stop Solution      1 x 3.5 ml
Standard
Standard
  1
Standard
2
Standard
  3
Standard
4
Standard
  5
Concentration
(mIU/ml)
0.5  2  10  40  100 
  4
STORAGE
1.  Unopened  test  kits  should  be  stored  at  2-8°C  upon  receipt  and  the
microtiter plate should be kept in a sealed bag. The test kit may be used
throughout the expiration date of the kit. Refer  to  the package  label for
the expiration date.
2.  Opened  test  kits  will  remain  stable  until  the  expiring  date  shown,
provided it is stored as prescribed above.    
3.  A  microtiter  plate  reader  with  a  bandwidth  of  10  nm  or  less  and  an
optical  density  range  of  0-3  OD  or  greater  at  450nm  wavelength  is
acceptable for use in absorbance measurement.
REAGENT PREPARATION
1.  Bring all reagents and plate to room temperature for at least 30 minutes
before use. Unused wells need store at 2-8°C and av oid sunlight.
2.  Wash Buffer    If crystals have formed in the concentrate, warm to room
temperature and mix gently until the crystals have compley dissolved.
Dilute 15 ml of Wash Buffer Concentrate into deionized or distilled water
to prepare 300 ml of Wash Buffer.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear
eye, hand, face, and clothing protection when using this material.
OTHER SUPPLIES REQUIRED
  Microplate  reader  capable  of measuring  absorbance  at  450  nm, with
the correction wavelength set at 540 nm or 570 nm.
  Pipettes and pipette tips.
  Deionized or distilled water.
  Squirt bottle, manifold dispenser, or automated microplate washer. 
  5
SAMPLE COLLECTION AND STORAGE
  Serum    Use a serum separator  tube (SST) and allow samples  to clot
for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove
serum and assay  immediay or aliquot and store samples at  -20° C.
Avoid repeated freeze-thaw cycles.
  Plasma    Collect  plasma  using  citrate,  EDTA,  or  heparin  as  an
anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of
collection. Assay  immediay  or  aliquot  and  store  samples  at  -20°C.
Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is
recommended that all samples, standards, and controls be assayed in duplicate.
1.  Set a Blank well without any solution. Add 50µl of Standard or Sample
per well. Standard need test in duplicate.  
2.  Add 50µl of HRP-conjugate and 50µl of antibody to each well (not to
Blank well). Mix well and then incubate for 1 hour at 37°C.  
3.  Complete  remove  the  liquid.  Then  fill  each  well  with  Wash  Buffer
(about 200µl), stay  for 10 seconds and Spinning. Repeat  the process
for a  total of three washes. Complete removal of  liquid at each step  is
essential  to  good  performance.  After  the  last  wash,  remove  any
remaining Wash Buffer by aspirating or decanting. Invert the plate and
blot it against clean paper towels.
4.  Add 50µl of Substrate A and 50µl of Substrate B to each well, mix well.
Incubate  for 15 minutes at 37°C. Keeping  the plate  away  from drafts
and other temperature fluctuations in the dark. 
  6
5.  Add  50µl  of  Stop  Solution  to  each  well.  If  color  change  does  not
appear uniform, gently tap the plate to ensure thorough mixing.
6.  Determine  the optical density of each well within 10 minutes, using a
microplate reader set to 450 nm.
CALCULATION OF RESULTS
Average the duplicate readings for each standard, control, and sample and
subtract the average zero standard optical density. Create a standard curve
by reducing the data using computer software capable of generating a four
parameter  logistic  (4-PL) curve-fit. As an alternative, construct a standard
curve  by  plotting  the mean  absorbance  for  each  standard  on  the  y-axis
against the concentration on the x-axis and draw a best fit curve through the
points on  the graph. The data may be  linearized by plotting  the  log of  the
FSH concentrations versus  the  log of  the O.D. and  the best fit  line can be
determined  by  regression  analysis.  This  procedure  will  produce  an
adequate but less precise fit of the data. If samples have been diluted, the
concentration  read  from  the  standard  curve  must  be  multiplied  by  the
dilution factor.
LIMITATIONS OF THE PROCEDURE
  The kit should not be used beyond the expiration date on the kit label.
  Do not mix or substitute reagents with those from other lots or sources.
  It is important that the Calibrator Diluent selected for the standard curve
be consistent with the samples being assayed.
  If samples generate values higher than the highest standard, dilute the
samples with the appropriate Calibrator Diluent and repeat the assay.
  Any  variation  in  Standard  Diluent,  operator,  pipetting  technique,
washing  technique,  incubation  time  or  temperature,  and  kit  age  can
cause variation in binding. 
  7
  This assay  is designed  to eliminate  interference by soluble  receptors,
binding proteins, and other  factors present  in biological samples. Until
all  factors  have  been  tested  in  the  Quantikine  Immunoassay,  the
possibility of interference cannot be excluded.
TECHNICAL HINTS
  When mixing or reconstituting protein solutions, always avoid foaming.
  To avoid cross-contamination, change pipette tips between additions of
each standard  level, between sample additions, and between  reagent
additions. Also, use separate reservoirs for each reagent.
  When  using  an  automated  plate  washer,  adding  a  30  second  soak
period  following  the  addition  of wash  buffer,  and/or  rotating  the  plate
180 degrees between wash steps may improve assay precision.
  To  ensure  accurate  results,  proper  adhesion  of  plate  sealers  during
incubation steps is necessary.
  Substrate  Solution  should  remain  colorless  until  added  to  the  plate.
Keep Substrate Solution protected from light. Substrate Solution should
change from colorless to gradations of blue.
  Stop Solution  should be added  to  the plate  in  the  same order as  the
Substrate Solution. The color developed in the wells will turn from blue
to  yellow  upon  addition  of  the Stop Solution. Wells  that  are  green  in
color indicate that the Stop Solution has not mixed thoroughly with the
Substrate Solution.
 
 
 
 
 
  8
大 大大 大鼠促卵泡素 鼠促卵泡素 鼠促卵泡素 鼠促卵泡素(FSH)快速檢測(cè)試劑盒 快速檢測(cè)試劑盒 快速檢測(cè)試劑盒 快速檢測(cè)試劑盒
使用說(shuō)明書(shū) 使用說(shuō)明書(shū) 使用說(shuō)明書(shū) 使用說(shuō)明書(shū)
本試劑盒僅供研究使用 本試劑盒僅供研究使用 本試劑盒僅供研究使用 本試劑盒僅供研究使用
產(chǎn)品編號(hào) 產(chǎn)品編號(hào) 產(chǎn)品編號(hào) 產(chǎn)品編號(hào):CSB-E06869r
檢測(cè)范圍 檢測(cè)范圍 檢測(cè)范圍 檢測(cè)范圍: :: :0.5 mIU/ml-100 mIU/ml
zui低檢測(cè)限 zui低檢測(cè)限 zui低檢測(cè)限 zui低檢測(cè)限: :: :0.2 mIU/ml
特異性 特異性 特異性 特異性: :: :本試劑盒可同時(shí)檢測(cè)天然或重組的 FSH,,且與其他相關(guān)蛋白基本
無(wú)交叉反應(yīng),。
有效期 有效期 有效期 有效期:6 個(gè)月(2-8℃避光保存)
預(yù)期應(yīng)用 預(yù)期應(yīng)用 預(yù)期應(yīng)用 預(yù)期應(yīng)用: :: : ELISA法定量測(cè)定大鼠血清,血漿及其它相關(guān)生物液體中 FSH
含量,。
說(shuō)明 說(shuō)明 說(shuō)明 說(shuō)明  
1.  濃洗滌液低溫保存會(huì)有鹽析出,,稀釋時(shí)可在水浴中加溫助溶。  
2.  剛開(kāi)啟的酶聯(lián)板孔中可能會(huì)含有少許水樣物質(zhì),,此為正?,F(xiàn)象,不會(huì)對(duì)實(shí)
驗(yàn)結(jié)果造成任何影響,。
實(shí)驗(yàn)原理 實(shí)驗(yàn)原理 實(shí)驗(yàn)原理 實(shí)驗(yàn)原理
采用酶聯(lián)免疫法競(jìng)爭(zhēng)法檢測(cè) FSH含量,。首先用羊抗兔包被微孔板,制備
成固相二抗,,然后加入待測(cè)標(biāo)本,、辣根過(guò)氧化物酶標(biāo)記 FSH 以及抗 FSH 抗
體,使之形成包被二抗-抗 FSH抗體-FSH  (HRP)復(fù)合物,。經(jīng)顯色后在酶標(biāo)儀
測(cè)定吸光值(OD值),,通過(guò)計(jì)算機(jī)或作圖擬合濃度-吸光度曲線,反算出待測(cè)
標(biāo)本中 FSH含量,。 
  9
試劑盒組成及試劑配制 試劑盒組成及試劑配制 試劑盒組成及試劑配制 試劑盒組成及試劑配制  
1.  酶聯(lián)板 酶聯(lián)板 酶聯(lián)板 酶聯(lián)板(Assay plate ):                                                              一塊 (48孔)。   
2.  標(biāo)準(zhǔn)品 標(biāo)準(zhǔn)品 標(biāo)準(zhǔn)品 標(biāo)準(zhǔn)品 (Standard):                                                                     5×0.5ml/瓶,。 
Standard 1  Standard 2  Standard 3  Standard 4  Standard 5
0.5 mIU/ml  2mIU/ml  10 mIU/ml  40 mIU/ml  100 mIU/ml
3.  抗體 抗體 抗體 抗體( (( (antibody) )) ):                                                                          1×3ml/瓶,。 
4.  酶結(jié)合物 酶結(jié)合物 酶結(jié)合物 酶結(jié)合物( (( (HRP-conjugate) )) ):                                                      1×3ml/瓶。 
5.  顯色劑 顯色劑 顯色劑 顯色劑 A( (( (Substrate A): ): ): ):                                                          1×3.5ml/瓶,。 
6.  顯色劑 顯色劑 顯色劑 顯色劑 B( (( (Substrate B): ): ): ):                                                          1×3.5ml/瓶,。 
7.  濃洗滌液 濃洗滌液 濃洗滌液 濃洗滌液( (( (Wash Buffer) )) ):1×15ml/瓶,使用時(shí)每瓶用蒸餾水稀釋 20 倍,。   
8.  終止液 終止液 終止液 終止液( (( (Stop Solution) )) ):                                                          1×3.5ml/瓶,。 
需要而未提供的試劑和器材 需要而未提供的試劑和器材 需要而未提供的試劑和器材 需要而未提供的試劑和器材
1.  標(biāo)準(zhǔn)規(guī)格酶標(biāo)儀
2.  高速離心機(jī)
3.  電熱恒溫培養(yǎng)箱
4.  干凈的試管和 Eppendof 管
5.  系列可調(diào)節(jié)移液器及吸頭,一次檢測(cè)樣品較多時(shí),,用多通道移液器
6.  蒸餾水,,容量瓶等
標(biāo)本的采集及保存 標(biāo)本的采集及保存 標(biāo)本的采集及保存 標(biāo)本的采集及保存
1.  血清:全血標(biāo)本請(qǐng)于室溫放置 2 小時(shí)或 4℃過(guò)夜后于 1000 x g離心 20 分
鐘,取上清即可檢測(cè),,或?qū)?biāo)本放于-20℃或-80℃保存,,但應(yīng)避免反復(fù)凍
融。
2.  血漿:可用 EDTA 或肝素作為抗凝劑,,標(biāo)本采集后 30 分鐘內(nèi)于 2 - 8° C
1000 x g離心 15 分鐘,,或?qū)?biāo)本放于-20℃或-80℃保存,,但應(yīng)避免反復(fù)
凍融。
3.  細(xì)胞培養(yǎng)物上清或其它生物標(biāo)本:1000 x g離心 20 分鐘,,取上清即可檢
測(cè),,或?qū)?biāo)本放于-20℃或-80℃保存,但應(yīng)避免反復(fù)凍融,。
注 注注 注: :: :以上標(biāo)本置 以上標(biāo)本置 以上標(biāo)本置 以上標(biāo)本置 4℃ ℃℃ ℃保存應(yīng)小于 保存應(yīng)小于 保存應(yīng)小于 保存應(yīng)小于 1 周 周周 周,, ,,, ,,-20℃ ℃℃ ℃或 或或 或-80℃ ℃℃ ℃均應(yīng)密封保存 均應(yīng)密封保存 均應(yīng)密封保存 均應(yīng)密封保存, ,,,, ,-20℃ ℃℃ ℃不應(yīng)超過(guò) 不應(yīng)超過(guò) 不應(yīng)超過(guò) 不應(yīng)超過(guò) 1 個(gè) 個(gè)個(gè) 個(gè)
月 月月 月,, ,,, ,,-80℃ ℃℃ ℃不應(yīng)超過(guò) 不應(yīng)超過(guò) 不應(yīng)超過(guò) 不應(yīng)超過(guò) 2 個(gè)月 個(gè)月 個(gè)月 個(gè)月,; ;,; ,;標(biāo)本溶血會(huì)影響zui后檢測(cè)結(jié)果 標(biāo)本溶血會(huì)影響zui后檢測(cè)結(jié)果 標(biāo)本溶血會(huì)影響zui后檢測(cè)結(jié)果 標(biāo)本溶血會(huì)影響zui后檢測(cè)結(jié)果, ,,,, ,因此溶血標(biāo)本不宜進(jìn)行此 因此溶血標(biāo)本不宜進(jìn)行此 因此溶血標(biāo)本不宜進(jìn)行此 因此溶血標(biāo)本不宜進(jìn)行此
項(xiàng)檢測(cè) 項(xiàng)檢測(cè) 項(xiàng)檢測(cè) 項(xiàng)檢測(cè),。 ,。。 ,。 
  10
操作步驟 操作步驟 操作步驟 操作步驟
1.  將各種試劑至室溫〔18-25℃〕平衡半小時(shí),,取濃縮洗滌液,根據(jù)當(dāng)批檢
測(cè)數(shù)量,,用蒸餾水上 1:20 稀釋?zhuān)靹蚝髠溆谩?
2.  將酶標(biāo)板取出,,設(shè)一個(gè)空白對(duì)照孔、不加任何液體,;每個(gè)標(biāo)準(zhǔn)點(diǎn)依次各設(shè)
兩孔,,每孔加入相應(yīng)標(biāo)準(zhǔn)品 50ul;其余每個(gè)檢測(cè)孔直接加待測(cè)標(biāo)本 50ul,。   
3.  每孔加入酶結(jié)合物 50ul(空白對(duì)照孔除外),,再按同樣的順序每孔加入抗
體 50 ul,,充分混勻,貼上不干膠封片,,置 37℃溫育 1 小時(shí),。
4.  手工洗板,棄去孔內(nèi)液體,。洗滌液注滿各孔,,靜置 10 秒甩干,重復(fù)三次
后拍干,;洗板機(jī)洗板,,選擇洗滌三次程序,洗板后拍干,。
5.  每孔加顯色劑 A 液 50µl,,顯色劑 B 液 50µl,振蕩混勻后,,37℃避光顯色
15 分鐘,,每孔加終止液 50µl。
6.  用酶標(biāo)儀讀數(shù),,取波長(zhǎng) 450nm,,先用空白孔調(diào)零點(diǎn),然后測(cè)定各孔 OD
值,。
數(shù)據(jù)處理 數(shù)據(jù)處理 數(shù)據(jù)處理 數(shù)據(jù)處理
1.  手工作圖:用雙對(duì)數(shù)坐標(biāo)紙,,以標(biāo)準(zhǔn)品濃度為橫軸,以對(duì)應(yīng)的 0D值為縱
軸,,畫(huà)出平滑曲線或直線,,在曲線上按照待測(cè)血清 OD值找到對(duì)應(yīng)的濃度
值。
2.  計(jì)算機(jī):使用線性擬合功能,,應(yīng)將標(biāo)準(zhǔn)品 S1-S5 的濃度取對(duì)數(shù)(Log(濃
度))作為 X,將對(duì)應(yīng)的 OD值減去空白對(duì)照孔 OD值后取對(duì)數(shù)(Log(OD
值-NSB))作為 Y,,進(jìn)行線性擬合,。再?gòu)臄M合線上計(jì)算出待測(cè)血清濃度。
注意事項(xiàng) 注意事項(xiàng) 注意事項(xiàng) 注意事項(xiàng)
1.  從冷藏環(huán)境中取出的試劑盒內(nèi)全部瓶裝試劑及所需預(yù)包被板條應(yīng)置室溫
(18-25℃)平衡 30 分鐘后方可使用,,余者應(yīng)及時(shí)封好口,,放回 2-8℃中避
光保存,以備后用,。
2.  使用前試劑應(yīng)搖勻,。
3.  結(jié)果判斷須在反應(yīng)終止后 10 分鐘內(nèi)完成。
4.  不同批號(hào)的試劑不可混用,。
5.  加樣時(shí)應(yīng)注意避免所用各試劑及樣品之間的交又污染,。
6.  操作時(shí),,試劑盒內(nèi)每種試劑各使用一個(gè)吸頭,每一種標(biāo)準(zhǔn)品使用一個(gè)吸頭,,
每一個(gè)樣品各使用一個(gè)吸頭,,吸頭一次性使用。        
   
 
 
 

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