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三级黄,色曰本学生

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大鼠透明质酸(HA)酶联免疫分析ELISA试剂盒使用说明书本试剂盒仅供研究使用—北京奇松生物科技有限公司http://www.shiyanshiji.com产品编号:QS42052预期应用ELISA法定量测定大鼠血清、血浆以及蛋清和蛋黄中透明质酸(HA)含量。实验原理本试剂盒应用双抗体夹心酶标免疫分析法测定标本中透明质酸水平。用纯化的透明质酸结合蛋白(HABP)包被微孔板,制成固相抗体,往包被单抗的微孔中依次加入透明质酸、生物素化的抗大鼠透明质酸结合蛋白(HABP)、HRP标记的亲和素,经过彻底洗涤后用底物TMB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成最终的黄色。颜色的深浅和样品中的透明质酸呈正相关。用酶标仪在450nm波长下测定吸光度(OD值),计算样品浓度。试剂盒组成及试剂配制1.酶联板:一块(96孔)标准品(冻干品):2瓶,每瓶临用前以样品稀释液稀释至1ml,盖好后静置10分钟以上,然后反复颠倒/搓动以助溶解,其浓度为100 nmol/L,做系列倍比稀释后,分别稀释成100 nmol/L,50 nmol/L ,25 nmol/L,12.5 nmol/L,6.25 nmol/L,3.12 nmol/L,1.56 nmol/L,其原液直接作为最高标准浓度,样品稀释液直接作为标准浓度0 nmol/L,临用前15分钟内配制。如配制50 nmol/L标准品:取0.5ml 100 nmol/L的上述标准品加入含有0.5ml样品稀释液的Eppendorf管中,混匀即可,其余浓度以此类推。2.样品稀释液:1×20ml/瓶。3.检测稀释液A:1×10ml/瓶。4.检测稀释液B:1×10ml/瓶。5.检测溶液A:1×120ul/瓶(1:100)临用前以检测稀释液A1:100稀释,稀释前根据预先计算好的每次实验所需的总量配制(每孔100ul),实际配制时应多配制0.1-0.2ml。如1ul 检测溶液A加99ul检测稀释液A的比例配制,轻轻混匀,在使用前一小时内配制。6.检测溶液B:1×120ul/瓶(1:100)临用前以检测稀释液B1:100稀释。稀释方法同检测溶液A。7.底物溶液:1×10ml/瓶。8.浓洗涤液:1×30ml/瓶,使用时每瓶用蒸馏水稀释25倍。9.终止液:1×10ml/瓶(2N H2SO4)。标本的采集及保存1.血清:标本请于室温放置2小时或4℃过夜后于1000 x g离心20分钟,取上清即可检测,或将标本放于-20℃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 x g离心15分钟,或将标本放于-20℃保存,但应避免反复冻融。3. 大鼠蛋:分离蛋清和蛋黄,处理后的标本放于-20℃保存,但应避免反复冻融。,注:标本溶血会影响最后检测结果,因此溶血标本不宜进行此项检测。操作步骤实验开始前,请提前配置好所有试剂,试剂或样品稀释时,均需混匀,混匀时尽量避免起泡。每次检测都应该做标准曲线。如样品浓度过高时,用样品稀释液进行稀释,以使样品符合试剂盒的检测范围。1.加样:分别设空白孔、标准孔、待测样品孔。空白孔加样品稀释液100ul,余孔分别加标准品或待测样品100ul,注意不要有气泡,加样将样品加于酶标板孔底部,尽量不触及孔壁,轻轻晃动混匀,酶标板加上盖或覆膜,37℃反应120分钟。为保证实验结果有效性,每次实验请使用新的标准品溶液。2.弃去液体,甩干,不用洗涤。每孔加检测溶液A工作液100ul(取1ul检测溶液A加99ul检测稀释液A的比例配制,轻轻混匀,在使用前一小时内配制),37℃,60分钟。3.温育60分钟后,弃去孔内液体,甩干,洗板3次,每次浸泡1-2分钟,350ul/每孔,甩干。4.每孔加检测溶液B工作液(同检测A工作液)100ul,37℃,60分钟。5.温育60分钟后,弃去孔内液体,甩干,洗板5次,每次浸泡1-2分钟,350ul/每孔,甩干。6.依序每孔加底物溶液90ul,37℃避光显色(30分钟内,此时肉眼可见标准品的前3-4孔有明显的梯度兰色,后3-4孔梯度不明显,即可终止)。7.依序每孔加终止溶液50ul,终止反应(此时蓝色立转黄色)。终止液的加入顺序应尽量与底物液的加入顺序相同。为了保证实验结果的准确性,底物反应时间到后应尽快加入终止液。8.用酶联仪在450nm波长依序测量各孔的光密度(OD值)。在加终止液后15分钟以内进行检测。注:1.每次实验留一孔作为空白调零孔,该孔不加任何试剂,只是最后加底物溶液及2NH2SO4。测量时先用此孔调OD值至零。2.为防止样品蒸发,试验时将反应板放于铺有湿布的密闭盒内,酶标板加上盖或覆膜。3. 未使用完的酶标板或者试剂,请于2-8℃保存。标准品、检测溶液A工作液、检测溶液B工作液请依据所需的量配置使用。请勿重复使用已稀释过的标准品、检测溶液A工作液或检测溶液B工作液。4. 建议检测样品时均设双孔测定,以保证检测结果的准确性。洗板方法手工洗板方法:吸去(不可触及板壁)或甩掉酶标板内的液体;在实验台上铺垫几层吸水纸,酶标板朝下用力拍几次;将推荐的洗涤缓冲液至少0.3ml注入孔内,浸泡1-2分钟,根据需要,重复此过程数次。自动洗板:如果有自动洗板机,应在熟练使用后再用到正式实验过程中。特异性本试剂盒可同时检测重组或天然的大鼠透明质酸,且与其它相关蛋白无交叉反应。计算以标准物的浓度为横坐标(对数坐标),OD值为纵坐标(普通坐标),在半对数坐标纸上绘出标准曲线,根据样品的OD值由标准曲线查出相应的浓度;再乘以稀释倍数;或用标准物的浓度与OD值计算出标准曲线的直线回归方程式,将样品的OD值代入方程式,计算出样品浓度,再乘以稀释倍数,即为样品的实际浓度。注意事项1.洗涤过程非常重要,不充分的洗涤易造成假阳性。2.一次加样时间最好控制在5分钟内,如标本数量多,推荐使用排枪加样。3.请每次测定的同时做标准曲线,最好做复孔。4.如标本中待测物质含量过高,请先稀释后再测定,计算时请最后乘以稀释倍数。5.在配制标准品、检测溶液工作液时,请以相应的稀释液配制,不能混淆。6.底物请避光保存。检测范围:1.56 nmol/L -100 nmol/L说明1.试剂盒保存:-20℃(较长时间不用时);2-8℃(频繁使用时)。2.有效期:6个月3.浓洗涤液会有盐析出,稀释时可在水浴中加温助溶。4.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。5.中、英文说明书可能会有不一致之处,请以英文说明书为准。英文版Rat Hyaluronic acid (HA) ELISA kitCatalog No. QS42052(96 tests)Intended useThis immunoassay kit allows for the specific measurement of Rat HA concentrations in serum and plasma.IntroductionHyaluronic Acid (HA), also called hyaluronate or hyaluronan, is a mucopolysaccharide widely distributed throughout the body. HA is produced mainly by fibroblasts and other specialized connective tissue cells. As a free molecule, HA can be found in the plasma and synovial fluid. HA is quickly removed from circulation by specific receptors present in sinusoidal cells (SEC) of the liver; the estimated half-life in plasma is 5-6 minutes.T est principleThis assay employs the quantitative sandwich enzyme immunoassay technique. A Hyaluronic Acid binding protein (HABP) specific for HA has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any HA present is bound. An enzyme-linked HABP specific for HA is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of HA bound in the initial step. The color development is stopped and the intensity of the color is measured.Materials and componentsReagent QuantityAssay plate 1Standard 2Sample Diluent 1 x 20mlAssay Diluent A 1 x 10mlAssay Diluent B 1 x 10mlDetection Reagent A 1 x 120ulDetection Reagent B 1 x 120ulWash Buffer 1 x 30ml(25 x concentrate)Substrate 1 x 10mlStop Solution 1 x 10mlSample collection and storageSerum- Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20° C.Plasma- Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for15 minutes at 1000 x g at 2 - 8° C within 30 minutes of collection. Store samples at ≤-20° C. A void repeated freeze-thaw cycles.Note: Citrate plasma has not been validated for use in this assay.Limitations of the procedureFOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.1. The kit should not be used beyond the expiration date on the kit label.2. Do not mix or substitute reagents with those from other lots or sources.3. If samples generate values higher than the highest standard, further dilute the sampleswith the Assay 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.4. This assay is designed to eliminate interference by soluble receptors, ligands, 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.Reagent preparationBring all reagents to room temperature before use.Wash Buffer - If crystals have formed in the concentrate, warm 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 500 ml of Wash Buffer.Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 100 nmol/L. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (100 nmol/L). The Sample Diluent serves as the zero standard (0 nmol/L).Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100),respectively.Assay procedureAllow all reagents to reach room temperature. Arrange and label required number of strips.1. Prepare all reagents, working standards and samples as directed in the previous sections.2. Add 100 uL of Standard, Control, or sample* per well. Cover with the adhesive strip. Incubate for2 hours at 37° C.3. Remove the liquid of each well, don’t wash.4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C. Detection ReagentA may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. 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. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37° C.7. Repeat the aspiration/wash as in step 5.8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature.Protect from light.9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap theplate to ensure thorough mixing.10. Determine the optical density of each well within 30 minutes, using a microplate reader setto 450 nm.SpecificityThis assay recognizes recombinant and natural Rat HA. No significant cross-reactivity or interference was observed.Important Note:1.The wash procedure is critical. Insufficient washing will result in poor precision and falselyelevated absorbance readings.2.It is recommended that no more than 32 wells be used for each assay run if manual pipetting isused since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.3.Duplication of all standards and specimens, although not required, is recommended.4.When mixing or reconstituting protein solutions, always avoid foaming.5.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.6.To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.Calculation of resultsA verage 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 softwarecapable 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 againstthe 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 HA 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.Storage of test kits and instrumentation1. Unopened test kits should be stored at 2-8 C upon receipt and the microtiter plate should be kept ina sealed bag with desiccants to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit (six months from the date of manufacture). 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 prescribedabove.3. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.PrecautionThe Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.大鼠透明质酸(HA)酶联免疫分析ELISA试剂盒使用说明书本试剂盒仅供研究使用—北京奇松生物科技有限公司http://www.shiyanshiji.com产品编号:QS42052预期应用ELISA法定量测定大鼠血清、血浆以及蛋清和蛋黄中透明质酸(HA)含量。实验原理本试剂盒应用双抗体夹心酶标免疫分析法测定标本中透明质酸水平。用纯化的透明质酸结合蛋白(HABP)包被微孔板,制成固相抗体,往包被单抗的微孔中依次加入透明质酸、生物素化的抗大鼠透明质酸结合蛋白(HABP)、HRP标记的亲和素,经过彻底洗涤后用底物TMB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成最终的黄色。颜色的深浅和样品中的透明质酸呈正相关。用酶标仪在450nm波长下测定吸光度(OD值),计算样品浓度。试剂盒组成及试剂配制1.酶联板:一块(96孔)标准品(冻干品):2瓶,每瓶临用前以样品稀释液稀释至1ml,盖好后静置10分钟以上,然后反复颠倒/搓动以助溶解,其浓度为100 nmol/L,做系列倍比稀释后,分别稀释成100 nmol/L,50 nmol/L ,25 nmol/L,12.5 nmol/L,6.25 nmol/L,3.12 nmol/L,1.56 nmol/L,其原液直接作为最高标准浓度,样品稀释液直接作为标准浓度0 nmol/L,临用前15分钟内配制。如配制50 nmol/L标准品:取0.5ml 100 nmol/L的上述标准品加入含有0.5ml样品稀释液的Eppendorf管中,混匀即可,其余浓度以此类推。2.样品稀释液:1×20ml/瓶。3.检测稀释液A:1×10ml/瓶。4.检测稀释液B:1×10ml/瓶。5.检测溶液A:1×120ul/瓶(1:100)临用前以检测稀释液A1:100稀释,稀释前根据预先计算好的每次实验所需的总量配制(每孔100ul),实际配制时应多配制0.1-0.2ml。如1ul 检测溶液A加99ul检测稀释液A的比例配制,轻轻混匀,在使用前一小时内配制。6.检测溶液B:1×120ul/瓶(1:100)临用前以检测稀释液B1:100稀释。稀释方法同检测溶液A。7.底物溶液:1×10ml/瓶。8.浓洗涤液:1×30ml/瓶,使用时每瓶用蒸馏水稀释25倍。9.终止液:1×10ml/瓶(2N H2SO4)。标本的采集及保存1.血清:标本请于室温放置2小时或4℃过夜后于1000 x g离心20分钟,取上清即可检测,或将标本放于-20℃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 x g离心15分钟,或将标本放于-20℃保存,但应避免反复冻融。3. 大鼠蛋:分离蛋清和蛋黄,处理后的标本放于-20℃保存,但应避免反复冻融。,注:标本溶血会影响最后检测结果,因此溶血标本不宜进行此项检测。操作步骤实验开始前,请提前配置好所有试剂,试剂或样品稀释时,均需混匀,混匀时尽量避免起泡。每次检测都应该做标准曲线。如样品浓度过高时,用样品稀释液进行稀释,以使样品符合试剂盒的检测范围。1.加样:分别设空白孔、标准孔、待测样品孔。空白孔加样品稀释液100ul,余孔分别加标准品或待测样品100ul,注意不要有气泡,加样将样品加于酶标板孔底部,尽量不触及孔壁,轻轻晃动混匀,酶标板加上盖或覆膜,37℃反应120分钟。为保证实验结果有效性,每次实验请使用新的标准品溶液。2.弃去液体,甩干,不用洗涤。每孔加检测溶液A工作液100ul(取1ul检测溶液A加99ul检测稀释液A的比例配制,轻轻混匀,在使用前一小时内配制),37℃,60分钟。3.温育60分钟后,弃去孔内液体,甩干,洗板3次,每次浸泡1-2分钟,350ul/每孔,甩干。4.每孔加检测溶液B工作液(同检测A工作液)100ul,37℃,60分钟。5.温育60分钟后,弃去孔内液体,甩干,洗板5次,每次浸泡1-2分钟,350ul/每孔,甩干。6.依序每孔加底物溶液90ul,37℃避光显色(30分钟内,此时肉眼可见标准品的前3-4孔有明显的梯度兰色,后3-4孔梯度不明显,即可终止)。7.依序每孔加终止溶液50ul,终止反应(此时蓝色立转黄色)。终止液的加入顺序应尽量与底物液的加入顺序相同。为了保证实验结果的准确性,底物反应时间到后应尽快加入终止液。8.用酶联仪在450nm波长依序测量各孔的光密度(OD值)。在加终止液后15分钟以内进行检测。注:1.每次实验留一孔作为空白调零孔,该孔不加任何试剂,只是最后加底物溶液及2NH2SO4。测量时先用此孔调OD值至零。2.为防止样品蒸发,试验时将反应板放于铺有湿布的密闭盒内,酶标板加上盖或覆膜。3. 未使用完的酶标板或者试剂,请于2-8℃保存。标准品、检测溶液A工作液、检测溶液B工作液请依据所需的量配置使用。请勿重复使用已稀释过的标准品、检测溶液A工作液或检测溶液B工作液。4. 建议检测样品时均设双孔测定,以保证检测结果的准确性。洗板方法手工洗板方法:吸去(不可触及板壁)或甩掉酶标板内的液体;在实验台上铺垫几层吸水纸,酶标板朝下用力拍几次;将推荐的洗涤缓冲液至少0.3ml注入孔内,浸泡1-2分钟,根据需要,重复此过程数次。自动洗板:如果有自动洗板机,应在熟练使用后再用到正式实验过程中。特异性本试剂盒可同时检测重组或天然的大鼠透明质酸,且与其它相关蛋白无交叉反应。计算以标准物的浓度为横坐标(对数坐标),OD值为纵坐标(普通坐标),在半对数坐标纸上绘出标准曲线,根据样品的OD值由标准曲线查出相应的浓度;再乘以稀释倍数;或用标准物的浓度与OD值计算出标准曲线的直线回归方程式,将样品的OD值代入方程式,计算出样品浓度,再乘以稀释倍数,即为样品的实际浓度。注意事项1.洗涤过程非常重要,不充分的洗涤易造成假阳性。2.一次加样时间最好控制在5分钟内,如标本数量多,推荐使用排枪加样。3.请每次测定的同时做标准曲线,最好做复孔。4.如标本中待测物质含量过高,请先稀释后再测定,计算时请最后乘以稀释倍数。5.在配制标准品、检测溶液工作液时,请以相应的稀释液配制,不能混淆。6.底物请避光保存。检测范围:1.56 nmol/L -100 nmol/L说明1.试剂盒保存:-20℃(较长时间不用时);2-8℃(频繁使用时)。2.有效期:6个月3.浓洗涤液会有盐析出,稀释时可在水浴中加温助溶。4.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。5.中、英文说明书可能会有不一致之处,请以英文说明书为准。英文版Rat Hyaluronic acid (HA) ELISA kitCatalog No. QS42052(96 tests)Intended useThis immunoassay kit allows for the specific measurement of Rat HA concentrations in serum and plasma.IntroductionHyaluronic Acid (HA), also called hyaluronate or hyaluronan, is a mucopolysaccharide widely distributed throughout the body. HA is produced mainly by fibroblasts and other specialized connective tissue cells. As a free molecule, HA can be found in the plasma and synovial fluid. HA is quickly removed from circulation by specific receptors present in sinusoidal cells (SEC) of the liver; the estimated half-life in plasma is 5-6 minutes.T est principleThis assay employs the quantitative sandwich enzyme immunoassay technique. A Hyaluronic Acid binding protein (HABP) specific for HA has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any HA present is bound. An enzyme-linked HABP specific for HA is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of HA bound in the initial step. The color development is stopped and the intensity of the color is measured.Materials and componentsReagent QuantityAssay plate 1Standard 2Sample Diluent 1 x 20mlAssay Diluent A 1 x 10mlAssay Diluent B 1 x 10mlDetection Reagent A 1 x 120ulDetection Reagent B 1 x 120ulWash Buffer 1 x 30ml(25 x concentrate)Substrate 1 x 10mlStop Solution 1 x 10mlSample collection and storageSerum- Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20° C.Plasma- Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for15 minutes at 1000 x g at 2 - 8° C within 30 minutes of collection. Store samples at ≤-20° C. A void repeated freeze-thaw cycles.Note: Citrate plasma has not been validated for use in this assay.Limitations of the procedureFOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.1. The kit should not be used beyond the expiration date on the kit label.2. Do not mix or substitute reagents with those from other lots or sources.3. If samples generate values higher than the highest standard, further dilute the sampleswith the Assay 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.4. This assay is designed to eliminate interference by soluble receptors, ligands, 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.Reagent preparationBring all reagents to room temperature before use.Wash Buffer - If crystals have formed in the concentrate, warm 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 500 ml of Wash Buffer.Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 100 nmol/L. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (100 nmol/L). The Sample Diluent serves as the zero standard (0 nmol/L).Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100),respectively.Assay procedureAllow all reagents to reach room temperature. Arrange and label required number of strips.1. Prepare all reagents, working standards and samples as directed in the previous sections.2. Add 100 uL of Standard, Control, or sample* per well. Cover with the adhesive strip. Incubate for2 hours at 37° C.3. Remove the liquid of each well, don’t wash.4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C. Detection ReagentA may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. 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. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37° C.7. Repeat the aspiration/wash as in step 5.8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature.Protect from light.9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap theplate to ensure thorough mixing.10. Determine the optical density of each well within 30 minutes, using a microplate reader setto 450 nm.SpecificityThis assay recognizes recombinant and natural Rat HA. No significant cross-reactivity or interference was observed.Important Note:1.The wash procedure is critical. Insufficient washing will result in poor precision and falselyelevated absorbance readings.2.It is recommended that no more than 32 wells be used for each assay run if manual pipetting isused since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.3.Duplication of all standards and specimens, although not required, is recommended.4.When mixing or reconstituting protein solutions, always avoid foaming.5.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.6.To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.Calculation of resultsA verage 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 softwarecapable 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 againstthe 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 HA 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.Storage of test kits and instrumentation1. Unopened test kits should be stored at 2-8 C upon receipt and the microtiter plate should be kept ina sealed bag with desiccants to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit (six months from the date of manufacture). 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 prescribedabove.3. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.PrecautionThe Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

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