CSB-E09815h

Transcription

1 Human growth hormone releasing peptide-ghrelin (GHRP-Ghrelin) ELISA Kit Catalog No. CSB-E09815h (96T) This immunoassay kit allows for the in vitro quantitative determination of human GHRP-Ghrelin 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

2 INTRODUCTION Ghrelin, a novel growth hormone releasing peptide is an acylated peptide that stimulates the release of growth hormone from pituitary. Ghrelin is synthesized as a preprohormone, then proteolytically processed to yield a 28-amino acid peptide. An interesting and unique modification is imposed on the hormone during synthesis in the form of an n-octanoic acid bound to one of its amino acids; this modification is necessary for biologic activity. Synthesis of ghrelin occurs predominantly in epithelial cells lining the fundus of the stomach, with smaller amounts produced in the placenta, kidney, pituitary and hypothalamus.ghrelin has been implicated in regulation of hunger and long-term weight gain/loss. Ghrelin is the endogenous ligand for the growth hormone secretagogue (GHS) receptor and has potent growth hormone-releasing activity. This peptide may constitute a new regulatory mechanism for GH-release. It is conceivable that GHRP-Ghrelin may also have other functions in some tissues in addition to pituitary, because the GHS receptor is expressed in heart, lung, pancreas, intestine, and adipose tissue. Ghrelin concentrations in blood are reduced in obese humans compared to lean control subjects, but whether this is cause or effect is not defined. Patients with anorexia nervosa have higher than normal plasma ghrelin levels, which decrease if weight gain occurs. PRINCIPLE OF THE ASSAY The microtiter plate provided in this kit has been pre-coated with biotin-bsa 2

3 and Avidin. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated GHRP-Ghrelin antibody and incubated. Then Horseradish Peroxidase (HRP)-conjugated antibody preparation specific for GHRP-Ghrelin 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 GHRP-Ghrelin in the samples is then determined by comparing the O.D. of the samples to the standard curve. DETECTION RANGE 50 pg/ml-1000 pg/ml. The standard curve concentrations used for the ELISA s were 1000 pg/ml, 500 pg/ml, 200 pg/ml, 100 pg/ml,50 pg/ml. SPECIFICITY This assay recognizes human GHRP-Ghrelin. No significant cross-reactivity or interference was observed. SENSITIVITY The minimum detectable dose of human GHRP-Ghrelin is typically less than 20 pg/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. 3

4 MATERIALS PROVIDED Reagent Quantity Assay plate 1 Standards (S1-S5) 5 x 1 ml Biotin-antibody 1 x 6 ml HRP-conjugate 1 x 10 ml Wash Buffer 1 x 20 ml (10 concentrate) Substrate A 1 x 7 ml Substrate B 1 x 7 ml Stop Solution 1 x 7 ml Standard Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Concentration (pg/ml) 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. 4

5 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. 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. 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. 5

6 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. 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 C. Keeping the pla te 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 6

7 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 GHRP-Ghrelin 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. 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. 7

8 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

9 人生长激素释放肽 ghrelin(ghrp-ghrelin) 酶联免疫分析试剂盒使用说明书本试剂盒仅供研究使用 产品编号 产品编号 :CSB-E09815h 检测范围 检测范围 :50 pg/ml 1000 pg/ml 最低检测限 最低检测限 :20 pg/ml 特异性 : 本试剂盒可检测人 GHRP-Ghrelin, 且与其他相关蛋白无交叉反 应 有效期 有效期 :6 个月 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 GHRP-Ghrelin 含量 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 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 GHRP-Ghrelin 的标准品 标本加入微孔板中, 使其与生物素标记的 GHRP-Ghrelin 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 GHRP-Ghrelin 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 9

10 试剂盒组成及试剂配制 1. 酶联板 (Assay plate ): 一块 (96 孔 ) 2. 标准品 (Standard): 5 1ml/ 瓶 Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 50 pg/ml 100 pg/ml 200 pg/ml 500 pg/ml 1000 pg/ml 3. 酶结合物 (HRP-Conjugate): 1 10ml/ 瓶 4. 生物素抗体 (Biotin-antibody) 1 6ml/ 瓶 5. 底物溶液 A(Substrate A): 1 7ml/ 瓶 6. 底物溶液 B(Substrate B): 1 7ml/ 瓶 7. 浓洗涤液 (Wash Buffer): ):): 1 20ml/ 瓶使用时每瓶用蒸馏水溶解到 200ml 8. 终止液 (Stop Solution): 1 7ml/ 瓶 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 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 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 : 以上标本置 4 保存应小于 1 周,-20 或 -80 均应密封保存,-20 不应超过 1 个月,-80 不应超过 2 个月 ; 标本溶血会影响最后检测结果, 因此溶血标本不宜进行检测 10

11 操作步骤 1. 将各种试剂至室温 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 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 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 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 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 11

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