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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.
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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
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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.
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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)
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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 completely
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.
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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 immediately 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 immediately 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.
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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.
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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.
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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.
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人生长激素人生长激素(GH)酶联免疫分析酶联免疫分析
人生长激素人生长激素 酶联免疫分析酶联免疫分析
试剂盒使用说明书试剂盒使用说明书
试剂盒使用说明书试剂盒使用说明书
本试剂盒仅供研究使用本试剂盒仅供研究使用
本试剂盒仅供研究使用本试剂盒仅供研究使用
产品编号产品编号：：CSB-E04567h
产品编号产品编号：：
检测范围检测范围：：2.5 ng/ml – 50 ng/ml
检测范围检测范围：：
最低检测限最低检测限：：1 ng/ml
最低检测限最低检测限：：
特异性特异性：：本试剂盒可检测人HGH，且与其他相关蛋白无交叉反应。
特异性特异性：：
有效期有效期：：6 个月
有效期有效期：：
预期应用预期应用：：ELISA 法定量测定人血清、血浆或其它相关生物液体中HGH
预期应用预期应用：：
含量。
说明说明
说明说明
1. 浓洗涤液低温保存会有盐析出，稀释时可在水浴中加温助溶。
2. 刚开启的酶联板孔中可能会含有少许水样物质，此为正常现象，不会对实
验结果造成任何影响。
实验原理实验原理
实验原理实验原理
用生物素化牛血清白蛋白和亲和素包被微孔板，制成固相载体，将已知
浓度的HGH 的标准品、标本加入微孔板中，使其与生物素标记的HGH 抗体
同时温育，洗涤后，加入辣根过氧化物酶标记的抗体，再经过温育和洗涤后
用底物显色。颜色的深浅和样品中的HGH 的浓度成比例关系。用酶标仪在
450nm 波长下测定吸光度 （OD 值），计算样品浓度。
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试剂盒组成及试剂配制试剂盒组成及试剂配制
试剂盒组成及试剂配制试剂盒组成及试剂配制
1. 酶联板酶联板(Assay plate )： 一块（96 孔）。
酶联板酶联板
2. 标准品标准品（Standard）： 5×1ml/瓶。
标准品标准品
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. 酶结合物酶结合物(HRP-Conjugate)： 1×10ml/瓶。
酶结合物酶结合物
4. 生物素抗体生物素抗体（（Biotin-antibody）） 1×6ml/瓶。
生物素抗体生物素抗体（（ ））
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. 浓洗涤液浓洗涤液（（Wash Buffer1×20ml/瓶 使用时每瓶用蒸馏水溶解到500ml。
浓洗涤液浓洗涤液（（
8. 终止液终止液（（Stop Solution）：） 1×7ml/瓶。
终止液终止液（（ ））
需要而未提供的试剂和器材需要而未提供的试剂和器材
需要而未提供的试剂和器材需要而未提供的试剂和器材
1. 标准规格酶标仪
2. 高速离心机
3. 电热恒温培养箱
4. 干净的试管和Eppendof 管
5. 系列可调节移液器及吸头，一次检测样品较多时，最好用多通道移液器
6. 蒸馏水，容量瓶等
标本的采集及保存标本的采集及保存
标本的采集及保存标本的采集及保存
1. 血清：全血标本请于室温放置2 小时或4℃过夜后于1000g 离心20 分钟，
取上清即可检测，或将标本放于-20℃或-80℃保存，但应避免反复冻融。
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2. 血浆：可用EDTA 或肝素作为抗凝剂，标本采集后30 分钟内于2 - 8°C
1000 g 离心15 分钟，或将标本放于-20℃或-80℃保存，但应避免反复冻
融。
注：注：以上标本置以上标本置4℃℃保存应小于保存应小于1 周，周，-20℃℃或或-80℃℃均应密封保存均应密封保存，，-20℃℃不应超过不应超过1 个个
注注：：以上标本置以上标本置 ℃℃保存应小于保存应小于 周周，， ℃℃或或 ℃℃均应密封保存均应密封保存，， ℃℃不应超过不应超过 个个
月，月，-80℃℃不应超过不应超过2 个月个月；标本溶；标本溶血会影响最后检测结果血会影响最后检测结果，因此溶血标本不宜进行检，因此溶血标本不宜进行检
月月，， ℃℃不应超过不应超过 个月个月；；标本溶标本溶血会影响最后检测结果血会影响最后检测结果，，因此溶血标本不宜进行检因此溶血标本不宜进行检
测。测。
测测。。
操作步骤操作步骤
操作步骤操作步骤
1. 将各种试剂至室温 〔18-25℃〕平衡半小时，取浓缩洗涤液，根据当批检
测数量，用蒸馏水上1：10 稀释，混匀后备用。
2. 将酶标板取出，分别向孔中加入50ul 标准品、标本，再分别加入50ul 生
物素化抗体，震动10-20 秒混匀，置37℃孵育30 分钟。
3. 手工洗板，弃去孔内液体。洗涤液注满各孔，静置10 秒甩干，重复三次
后拍干；洗板机洗板，选择洗涤三次程序，洗板后拍干。
4. 每孔加入100ul 酶结合物，震动10-20 秒混匀，置37℃孵育30 分钟。
5. 手工洗板，弃去孔内液体。洗涤液注满各孔，静置10 秒甩干，重复三次
后拍干；洗板机洗板，选择洗涤三次程序，洗板后拍干。
6. 每孔加显色剂A 液50μl，显色剂B 液50μl，振荡混匀后，18-25℃避光
显色15 分钟，每孔加终止液50μl。
7. 用酶标仪读数，取波长450nm，先用空白孔调零点，然后测定各孔OD
值。
数据处理数据处理
数据处理数据处理
1. 手工作图：用双对数坐标纸，以标准品浓度为横轴，以对应的0D 值为纵
轴，画出平滑曲线或直线，在曲线上按照待测血清OD 值找到对应的浓度
值。
2. 计算机：使用线性拟合功能，应将标准品S1-S5 的浓度取对数（Log(浓
度)）作为X，将对应的OD 值减去空白对照孔OD 值后取对数（Log(OD
值-NSB)）作为Y，进行线性拟合。再从拟合线上计算出待测血清浓度。
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注意事项注意事项
注意事项注意事项
1. 从冷藏环境中取出的试剂盒内全部瓶装试剂及所需预包被板条应置室温
（18-25℃）平衡30 分钟后方可使用，余者应及时封好口，放回2-8℃中避
光保存，以备后用。
2. 使用前试剂应摇匀。
3. 结果判断须在反应终止后10 分钟内完成。
4. 不同批号的试剂不可混用。
5. 加样时应注意避免所用各试剂及样品之间的交又污染。
6. 操作时，试剂盒内每种试剂各使用一个吸头，每一种标准品使用一个吸头，
每一个样品各使用一个吸头，吸头最好一次性使用。
7. 每次测试必须重新制作标准曲线，上次实验标准曲线不可重复使用。
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