您好, 歡迎來(lái)到化工儀器網(wǎng)! 登錄| 免費(fèi)注冊(cè)| 產(chǎn)品展廳| 收藏商鋪|
當(dāng)前位置:廈門(mén)慧嘉生物科技有限公司>>技術(shù)文章>>人生長(zhǎng)激素(GH)ELISA試劑盒
人生長(zhǎng)激素(GH)ELISA試劑盒
----------------------- Page 1-----------------------
Human Growth Hormone(HGH)
ELISA KIT
Catalog No. CSB-E04567h
(96T)
This immunoassay kit allows for the in vitro quantitative determination of human
HGH 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.
1
----------------------- Page 2-----------------------
INTRODUCTION
HGH is Human Growth Hormone, a natural hormone produced in the
pituitary gland of the brain. HGH is considered "the key" hormone because
it controls so many functions. It's responsible for youth, vitality, energy and
all of the health benefits we associate with youth. Dr. Daniel Rudman's
study in the New England Journal Of Medicine demonstrated the
remarkable ability to reverse the effects of aging upon the human body with
the employment of HGH - Human Growth Hormone! Due in part to his
efforts, Dr. Rudmans's study saw the effects of HGH upon overweight men
between the ages of 61 and 80 years of age.
HGH reduces body fat The men did not alter their personal habits of eating,
smoking, or exercise, yet with the consumption of HGH, they lost an
average of 14% of their body fat, while gaining an average of 8.8% lean
muscle mass. Their skin became firmer and they experienced a localized
increase in bone density. Over all, HgH appeared to reverse the effects of
aging by 10-20 years!!! HGH is prescribed and administered by a doctor in
the form of injections. But a nonprescription form is also available over the
counter and through mail order.
HGH promotes growth in children and plays an important role in adult
metabolism. The body secretes the hormone, in decreasing amounts,
throughout our lifetimes. The amount of hormone in the body can be
measured by levels of IGF-1 (Insulin Growth Factor). Growth hormone has
2
----------------------- Page 3-----------------------
a profound effect on all the cells of the body, more than any other hormone
because it is the cell generator.
HGH is the "master hormone" controlling many organs and body functions
and is directly responsible for stimulating tissue repair, cell replacement,
brain functions, and enzyme function! It’s human growth hormone that
grows the cells, bones, muscles, and organs, and it is the decreasing level
of human growth hormone after age 30 that slowly robs us of our "youth."
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with biotin-BSA
and Avidin. Standards or samples are then added to the appropriate
microtiter plate wells with a biotin-conjugated HGH antibody and incubated.
Then Horseradish Peroxidase (HRP)-conjugated antibody preparation
specific for HGH are added and incubated. Substrate solutions 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 HGH in the samples is then determined by comparing the
O.D. of the samples to the standard curve.
DETECTION RANGE
2.5 ng/ml-50 ng/ml. The standard curve concentrations used for the ELISA’s
were 50 ng/ml, 25ng/ml,10 ng/ml, 5 ng/ml,2.5 ng/ml.
3
----------------------- Page 4-----------------------
SPECIFICITY
This assay recognizes human HGH. No significant cross-reactivity or
interference was observed.
SENSITIVITY
The minimum detectable dose of human HGH is typically less than 1 ng/ml.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined
as the lowest protein concentration that could be differentiated from zero.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standards (S1-S4) 5x 1 ml
Biotin-antibody 1 x 6 ml
HRP-conjugate 1 x 10 ml
1 x 20 ml
Wash Buffer
(10×concentrate)
Substrate A 1 x 7 ml
Substrate B 1 x 7 ml
Stop Solution 1 x 7 ml
Standard Standard1 Standard2 Standard3 Standard4 Standard5
Concentration
2.5 5 10 25 50
(ng/ml)
4
----------------------- Page 5-----------------------
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
Bring all reagents to room temperature before use.
1. Wash Buffer If crystals have formed in the concentrate, warm up to
room temperature and mix gently until the crystals have compley
dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or
distilled water to prepare 200 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
----------------------- Page 6-----------------------
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. Add 50μl of Standard or Sample per well. Then add 50μl of
Biotin-antibody to each well. Standards need test in duplicate. Mix
well and then incubate for 30 min at 37°C.
2. 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.
6
----------------------- Page 7-----------------------
3. Add 50μl of HRP-conjugate to each well. Mix well and then incubate for
30 min at 37°C.
4. Wash the plate as before.
5. Add 50μl of Substrate A and 50μl of Substrate B to each well, mix well.
Incubate for 15 minutes at 18-25°C. Keeping the plate away from drafts
and other temperature fluctuations in the dark.
6. Add 50μl of Stop Solution to each well. If color change does not
appear uniform, gently tap the plate to ensure thorough mixing.
7. 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
HGH 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.
7
----------------------- Page 8-----------------------
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.
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.
8
----------------------- Page 9-----------------------
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.
9
----------------------- Page 10-----------------------
人生長(zhǎng)激素人生長(zhǎng)激素(GH)酶聯(lián)免疫分析酶聯(lián)免疫分析
人生長(zhǎng)激素人生長(zhǎng)激素 酶聯(lián)免疫分析酶聯(lián)免疫分析
試劑盒使用說(shuō)明書(shū)試劑盒使用說(shuō)明書(shū)
試劑盒使用說(shuō)明書(shū)試劑盒使用說(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)::CSB-E04567h
產(chǎn)品編號(hào)產(chǎn)品編號(hào)::
檢測(cè)范圍檢測(cè)范圍::2.5 ng/ml – 50 ng/ml
檢測(cè)范圍檢測(cè)范圍::
zui低檢測(cè)限zui低檢測(cè)限::1 ng/ml
zui低檢測(cè)限zui低檢測(cè)限::
特異性特異性::本試劑盒可檢測(cè)人HGH,且與其他相關(guān)蛋白無(wú)交叉反應(yīng)。
特異性特異性::
有效期有效期::6 個(gè)月
有效期有效期::
預(yù)期應(yīng)用預(yù)期應(yīng)用::ELISA 法定量測(cè)定人血清、血漿或其它相關(guān)生物液體中HGH
預(yù)期應(yīng)用預(yù)期應(yīng)用::
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
用生物素化牛血清白蛋白和親和素包被微孔板,制成固相載體,將已知
濃度的HGH 的標(biāo)準(zhǔn)品、標(biāo)本加入微孔板中,使其與生物素標(biāo)記的HGH 抗體
同時(shí)溫育,洗滌后,加入辣根過(guò)氧化物酶標(biāo)記的抗體,再經(jīng)過(guò)溫育和洗滌后
用底物顯色。顏色的深淺和樣品中的HGH 的濃度成比例關(guān)系。用酶標(biāo)儀在
450nm 波長(zhǎng)下測(cè)定吸光度 (OD 值),計(jì)算樣品濃度。
10
----------------------- Page 11-----------------------
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(Assay plate ): 一塊(96 孔)。
酶聯(lián)板酶聯(lián)板
2. 標(biāo)準(zhǔn)品標(biāo)準(zhǔn)品(Standard): 5×1ml/瓶。
標(biāo)準(zhǔn)品標(biāo)準(zhǔn)品
Standard 1 Standard 2 Standard 3 Standard 4 Standard 5
2.5 ng/ml 5 ng/ml 10ng/ml 25 ng/ml 50 ng/ml
3. 酶結(jié)合物酶結(jié)合物(HRP-Conjugate): 1×10ml/瓶。
酶結(jié)合物酶結(jié)合物
4. 生物素抗體生物素抗體((Biotin-antibody)) 1×6ml/瓶。
生物素抗體生物素抗體(( ))
5. 底物溶液底物溶液A ((Substrate A):) 1×7ml/瓶。
底物溶液底物溶液 (( ))
6. 底物溶液底物溶液B ((Substrate B):) 1×7ml/瓶。
底物溶液底物溶液 (( ))
7. 濃洗滌液濃洗滌液((Wash Buffer1×20ml/瓶 使用時(shí)每瓶用蒸餾水溶解到500ml。
濃洗滌液濃洗滌液((
8. 終止液終止液((Stop Solution):) 1×7ml/瓶。
終止液終止液(( ))
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1000g 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。
11
----------------------- Page 12-----------------------
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 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。
注:注: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1 周,周,-20℃℃或或-80℃℃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)1 個(gè)個(gè)
注注::以上標(biāo)本置以上標(biāo)本置 ℃℃保存應(yīng)小于保存應(yīng)小于 周周,, ℃℃或或 ℃℃均應(yīng)密封保存均應(yīng)密封保存,, ℃℃不應(yīng)超過(guò)不應(yīng)超過(guò) 個(gè)個(gè)
月,月,-80℃℃不應(yīng)超過(guò)不應(yīng)超過(guò)2 個(gè)月個(gè)月;標(biāo)本溶;標(biāo)本溶血會(huì)影響zui后檢測(cè)結(jié)果血會(huì)影響zui后檢測(cè)結(jié)果,因此溶血標(biāo)本不宜進(jìn)行檢,因此溶血標(biāo)本不宜進(jìn)行檢
月月,, ℃℃不應(yīng)超過(guò)不應(yīng)超過(guò) 個(gè)月個(gè)月;;標(biāo)本溶標(biāo)本溶血會(huì)影響zui后檢測(cè)結(jié)果血會(huì)影響zui后檢測(cè)結(jié)果,,因此溶血標(biāo)本不宜進(jìn)行檢因此溶血標(biāo)本不宜進(jìn)行檢
測(cè)。測(cè)。
測(cè)測(cè)。。
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1:10 稀釋?zhuān)靹蚝髠溆谩?/p>
2. 將酶標(biāo)板取出,分別向孔中加入50ul 標(biāo)準(zhǔn)品、標(biāo)本,再分別加入50ul 生
物素化抗體,震動(dòng)10-20 秒混勻,置37℃孵育30 分鐘。
3. 手工洗板,棄去孔內(nèi)液體。洗滌液注滿(mǎn)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。
4. 每孔加入100ul 酶結(jié)合物,震動(dòng)10-20 秒混勻,置37℃孵育30 分鐘。
5. 手工洗板,棄去孔內(nèi)液體。洗滌液注滿(mǎn)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。
6. 每孔加顯色劑A 液50μl,顯色劑B 液50μl,振蕩混勻后,18-25℃避光
顯色15 分鐘,每孔加終止液50μl。
7. 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(xiàn)或直線(xiàn),在曲線(xiàn)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(xiàn)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(xiàn)性擬合。再?gòu)臄M合線(xiàn)上計(jì)算出待測(cè)血清濃度。
12
----------------------- Page 13-----------------------
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。
7. 每次測(cè)試必須重新制作標(biāo)準(zhǔn)曲線(xiàn),上次實(shí)驗(yàn)標(biāo)準(zhǔn)曲線(xiàn)不可重復(fù)使用。
13
----------------------- Page 14-----------------------
Notes
14
----------------------- Page 15-----------------------
15
----------------------- Page 16-----------------------
16
----------------------- Page 17-----------------------
17
----------------------- Page 18-----------------------
18
----------------------- Page 19-----------------------
19
----------------------- Page 20-----------------------
20
請(qǐng)輸入賬號(hào)
請(qǐng)輸入密碼
請(qǐng)輸驗(yàn)證碼
以上信息由企業(yè)自行提供,信息內(nèi)容的真實(shí)性、準(zhǔn)確性和合法性由相關(guān)企業(yè)負(fù)責(zé),化工儀器網(wǎng)對(duì)此不承擔(dān)任何保證責(zé)任。
溫馨提示:為規(guī)避購(gòu)買(mǎi)風(fēng)險(xiǎn),建議您在購(gòu)買(mǎi)產(chǎn)品前務(wù)必確認(rèn)供應(yīng)商資質(zhì)及產(chǎn)品質(zhì)量。