大鼠(Rat)性激素结合球蛋白(SHBG)-NEWA

大鼠(Rat)性激素结合球蛋白(SHBG)-NEWA 分离。 本试剂盒只能用于科学研究,不得用于医学诊断 2. 血浆,EDTA、柠檬酸盐或肝素抗凝。3000转离心30分钟取上清。 大鼠，Rat，性激素结合球蛋白，SHBG， 3. 细胞上清液,3000转离心10分钟去除颗粒和聚合物。 ELISA检测试剂盒 使用说明 4. 组织匀浆,将组织加入适量生理盐水捣碎。3000转离心10分钟检测原理 取上清。 试剂盒采用双抗体一步夹心法酶联免疫吸附试验，ELISA，。往预5. 保存,如果样本收集后不及时检测,请按一次用量分装,冻存于先包被性激素结合球蛋白，SHBG，抗体的包被微孔中,依次加入标-20?,避免反复冻融,在室温下解冻并确保样品均匀地充分解冻。 本、品、HRP标记的检测抗体,经过温育并彻底洗涤。用底物TMB自备物品 显色,TMB在过氧化物酶的催化下转化成蓝色,并在酸的作用下转化1. 酶标仪，450nm， 成最终的黄色。颜色的深浅和样品中的性激素结合球蛋白，SHBG，2. 高精度加样器及枪头,0.5-10uL、2-20uL、20-200uL、200-1000uL 呈正相关。用酶标仪在450nm 波长下测定吸光度，OD 值，,计算样3. 37?恒温箱 品浓度。 操作注意事项 样品收集、处理及保存方法 1. 试剂盒保存在2-8?,使用前室温平衡20分钟。从冰箱取出的1. 血清,使用不含热原和内毒素的试管,操作过程中避免任何细胞浓缩洗涤液会有结晶,这属于正常现象,水浴加热使结晶完全溶解刺激,收集血液后,3000转离心10分钟将血清和红细胞迅速小心地后再使用。 2. 实验中不用的板条应立即放回自封袋中,密封，低温干燥，保存。 冲液加19份的蒸馏水。 3. 浓度为0的S0号标准品即可视为阴性对照或者空白,按照说明洗板方法 书操作时样本已经稀释5倍,最终结果乘以5才是样本实际浓度。 1. 手工洗板,甩尽孔内液体,每孔加满洗涤液,静置1min后甩尽4. 严格按照说明书中标明的时间、加液量及顺序进行温育操作。 孔内液体,在吸水纸上拍干,如此洗板5次。 5. 所有液体组分使用前充分摇匀。 2. 自动洗板机,每孔注入洗液350μL,浸泡1min,洗板5次。 试剂盒组成 操作步骤 1. 从室温平衡20min后的铝箔袋中取出所需板条,剩余板条用自封名称 96孔配置 48孔配置 备注 微孔酶标板 12孔×8条 12孔×4条 无 袋密封放回4?。 标准品 0.3mL*6管 0.3mL*6管 无 样本稀释液 6mL 3mL 无 2. 设置标准品孔和样本孔,标准品孔各加不同浓度的标准品50μL, 检测抗体-HRP 10mL 5mL 无 20×洗涤缓冲液 25mL 15mL 按说明书进行稀释 3. 样本孔先加待测样本10μL,再加样本稀释液40μL,空白孔不底物A 6mL 3mL 无 底物B 6mL 3mL 无 加。 终止液 6mL 3mL 无 封板膜 2张 2张 无 4. 除空白孔外,标准品孔和样本孔中每孔加入辣根过氧化物酶说明书 1份 1份 无 自封袋 1个 1个 无 ，HRP，标记的检测抗体100μL,用封板膜封住反应孔,37?水浴注,标准品，S0-S5，浓度依次为,0、7.5、15、30、60、120 nmol/L 锅或恒温箱温育60min。 试剂的准备 5. 弃去液体,吸水纸上拍干,每孔加满洗涤液,静置1min,甩去 20×洗涤缓冲液的稀释,蒸馏水按1,20稀释,即1份的20×洗涤缓 洗涤液,吸水纸上拍干,如此重复洗板5次，也可用洗板机洗板，。 6. 每孔加入底物A、B各50μL,37?避光孵育15min。 7. 每孔加入终止液50μL,15min内,在450nm波长处测定各孔的 OD值。 结果判断 绘制标准曲线,在Excel工作表中,以标准品浓度作横坐标,对应 OD值作纵坐标,绘制出标准品线性回归曲线,按曲线方程计算各样 本浓度值。 试剂盒性能 1. 准确性,标准品线性回归与预期浓度相关系数R值,大于等于 0.9900。 2. 灵敏度,最低检测浓度小于1.0 nmol/L。 3. 特异性,不与其它可溶性结构类似物交叉反应。 4. 重复性,板内、板间变异系数均小于15%。 5. 贮藏,2-8?,避光防潮保存。 6. 有效期,6个月 this SHBG ELISA Kit includes a set of calibration standards. The calibration 免责声明 standards are assayed at the same time as the samples and allow the operator to 1. 试剂盒仅供研究使用,不得用于临床实验或人体实验,否则所 produce a standard curve of Optical Density versus SHBG concentration. The 产生的一切后果,由实验者承担,本公司概不负责。 concentration of SHBG in the samples is then determined by comparing the O.D. of the samples to the standard curve. 2. 严格按照说明书操作,实验者违反说明书操作,后果由实验者 Sample collection and storages 承担。 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 FOR RESEARCH USE ONLY. freeze-thaw cycles NOT FOR USE IN DIAGNOSTIC PROCEDURES. 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 Rat sex hormone-binding globulin (SHBG) ELISA Kit samples at -20?or -80?. Avoid repeated freeze-thaw cycles. Cell culture supernates and other biological fluids - Remove particulates instruction by centrifugation and assay immediately or aliquot and store samples at -20?or -80?. Avoid repeated freeze-thaw cycles. Intended use Note: The samples shoule be centrifugated dequately and no hemolysis This SHBG ELISA kit is intended Laboratory for Research use only and is not for or granule was allowed. use in diagnostic or therapeutic procedures.The Stop Solution changes the color Materials required but not supplied 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 SHBG in the sample, 1. Standard microplate reader(450nm) User manual 1 1 2. Precision pipettes and Disposable pipette tips. Sealed bags 1 1 3. 37 ? incubator Note: Standard (S0 ? S5) concentration was followed by:0,7.5,15,30,60,120 nmol/L Precautions Reagent preparation 1. Do not substitute reagents from one kit to another. Standard, conjugate and 20×wash solution:Dilute with Distilled or deionized water 1:20. microplates are matched for optimal performance. Use only the reagents supplied by manufacturer. Assay procedure 2. Do not remove microplate from the storage bag until needed. Unused strips 1. Prepare all reagents before starting assay procedure. It is recommended that should be stored at 2-8?C in their pouch with the desiccant provided. all Standards and Samples be added in duplicate to the Microelisa Stripplate. 3. Mix all reagents before using. 2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to Remove all kit reagents from refrigerator and allow them to reach room temperature standard well. ( 20-25?C) 3. Add Sample: Add testing sample 10μl then add Sample Diluent 40μl to testing Materials supplied sample well; Blank well doesn’t add anyting. 4. Add 10Name 96 determinations 48 determinations 0μl of HRP-conjugate reagent to each well, cover with an adhesive strip Microelisa stripplate 12*8strips 12*4strips and incubate for 60 minutes at 37?C. Standard 0.3ml*6tubes 0.3ml*6tubes 5. Aspirate each well and wash, repeating the process four times for a total of five Sample Diluent 6.0ml 3.0ml HRP-Conjugate reagent 10.0ml 5.0ml washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle, 20X Wash solution 25ml 15ml manifold dispenser or autowasher. Complete removal of liquid at each step is Chromogen Solution A 6.0ml 3.0ml essential to good performance. After the last wash, remove any remaining Wash Chromogen Solution B 6.0ml 3.0ml Stop Solution 6.0ml 3.0ml Solution by aspirating or decanting. Invert the plate and blot it against clean paper Closure plate membrane 2 2 towels. 6. Add chromogen solution A 50μl and chromogen solution B 50μl to each well. 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 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 their own standard curve. from blue to yellow. If the color in the wells is green or the color change does not 5. The sensitivity by this assay is 1.0 nmol/L appear uniform, gently tap the plate to ensure thorough mixing. 6. Standard curve 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 Storage: 2-8?. validity: six months. intersection, draw a vertical line to the X-axis and read the corresponding concentration. FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!