扭转原肠胚形成同源物1ELISA试剂盒说明书

Rat IL-1beta ELISA KitCatalog NO.:RK00009version:2.0This package insert must be read in its entirety beforeusing this productIntroductionThe kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of IL-1beta in rat serum,plasma,cell culture supernatants.Principle of the AssayThis assay employs the quantitative sandwich enzyme immunoassay technique.An antibody specific for rat IL-1beta has been pre-coated onto a microplate.Standards and samples are pipetted into the wells and any IL-1beta present is bound by the immobilized antibody.After washing away any unbound substances, and then a detection antibody specific for IL-1beta is added to the wells and binds to the combination of capture antibody IL-1beta in sample.Following a wash to remove any unbound combination,and enzyme conjugate is added to the wells. Following incubation and wash steps,a substrate solution is added to the wells and color develops in proportion to the amount of IL-1beta bound in the initial step.The color development is stopped and the absorbance is measured.Material Provided&Storage ConditionsUnopened kits can be stored at2-8°C for1year,and opened products must be used within1month.Part Size Cat.No.Storage ofopened/reconstituted materialAntibody Coated Plate 8×12RM00064Put the unused slats backin the aluminum foil bagwith the desiccant andreseal them.They can bestored at2-8°C for1month.Standard Lyophilized 2vials RM00061It is not recommended touse again afterredissolving.Concentrated BiotinConjugate Antibody(100×)1×120ul RM00062Store at2-8°c for1month*Streptavidin-HRP Concentrated(100×)1×120ul RM00063Store at 2-8°c for 1month *Standard/SampleDiluent (R1)1×20mL RM00023Store at 2-8°c for 1month *Biotin-Conjugate AntibodyDiluent (R2)1×12mL RM00024Streptavidin-HRP Diluent(R3)1×12mL RM00025WashBuffer(20x)1×30mL RM00026TMB Substrate1×12mL RM00027Stop Solution1×6mL RM00028Plate Sealers4Strips Specification 1Other Supplies Required1.Microplate reader capable of measuring absorbance at450nm,with the correction wavelength set at630nm or570 nm.2.Pipettes and pipette tips.3.Deionized or distilled water.4.Squirt bottle,manifold dispenser,or automated microplatewasher.5.Incubator.6.Test tubes for dilution of standards and samples.Precautions1.Any variation in diluent,operator,pipetting technique,washing technique,incubation time or temperature,and kitage can cause variation in binding.2.Variations in sample collection,processing,and storagemay cause sample value differences.3.Reagents may be harmful,if ingested,rinse it with anexcess amount of tap water.4.Stop Solution contains strong acid.Wear eye,hand,andface protection.5.Please perform simple centrifugation to collect the liquidbefore use.6.Do not mix or substitute reagents with those from other lotsor other sources.7.Adequate mixing is particularly important for good result.Use a mini-vortexer at the lowest frequency.8.Mix the sample and all components in the kits adequately,and use clean plastic container to prepare all diluents.9.Both the sample and standard should be assayed in duplicate,and reagents should be added in sequence in accordance with the requirement of the specification.10.Reuse of dissolved standard is not recommended.11.The kit should not be used beyond the expiration date onthe kit label.12.The kit should be away from light when it is stored orincubated.13.To reduce the likelihood of blood-borne transmission ofinfectious agents,handle all serum,plasma,and other biological fluids in accordance with NCCLS regulations.14.To avoid cross contamination,please use disposablepipette tips.15.Please prepare all the kit components according to theSpecification.If the kits will be used several times, please seal the rest strips and preserve with desiccants.Do use up within2months.16.This assay is designed to eliminate interference by otherfactors present in biological samples.17.Until all factors have been tested in this assay,thepossibility of interference cannot be excluded.18.The48T kit is also suitable for the specification.Sample Collection&StorageThe sample collection and storage conditions listed below are intended as general guidelines.Sample stability has not been evaluated.Samples containing the correlated IgG as in this kit may interfere with this assay.Cell Culture Supernatant:Remove particulates by centrifugation. Assay immediately or aliquot and store samples at≤-20°C. Avoid repeated freeze-thaw cycles.Serum:Use a serum separator tube(SST)and allow samples to clot for30minutes at room temperature before centrifugation for15 minutes at1000x g.Remove serum and assay immediately or aliquot and store samples at≤-20°C.Avoid repeated freeze-thaw cycles.Plasma:Collect plasma using EDTA or Heparin as an anticoagulant. Centrifuge for15minutes at1000×g within30minutes of collection.Assay immediately or aliquot and store samples at ≤-20℃.Avoid repeated freeze-thaw cycles.(Note:Citrateplasma has not been validated for use in this assay.)Note:It is suggested that all samples in one experiment be collected at the same time of the day.Avoid hemolytic and hyperlipidemia sample for serum and plasma.Reagent PreparationBring all reagents to room temperature before use.If crystals have formed in the concentrate,Bring the reagent to room temperature and mix gently until the crystals have completely dissolved.Standard-Reconstitute the Standard Lyophilized with 1.0mL Standard/Sample Diluent(R1).This reconstitution produces a stock solution of8000pg/mL.Mix the standard to ensure complete reconstitution and allow the standard to sit for a minimum of 15minutes with gentle agitation prior to making dilutions. Use the8000pg/mL standard stock to produce a dilution series (below)with Standard/Sample Diluent(R1).Mix each tube thoroughly and change pipette tips between each transfer(recommended concentration for standard curve:4000,2000,1000,500,250,125,62.5,0pg/mL).Use diluted standards within 60minutes of preparation.Working Biotin Conjugate Antibody -Dilute 1:100of Concentrated Biotin Conjugate Antibody (100x)with Biotin-Conjugate Antibody Diluent (R2)before use,for example:Add 20μL of Concentrated Biotin Conjugate Antibody (100x)to 1980μL Biotin-Conjugate Antibody Diluent (R2)to prepare 2000μL Working Biotin Conjugate Antibody Buffer.Working Streptavidin-HRP -Dilute 1:100of Concentrated Streptavidin-HRP (100x)with Streptavidin-HRP Diluent (R3)before use,for example:Add 20μL of Concentrated Streptavidin-HRP (100x)to 1980μL Streptavidin-HRP Diluent (R3)Std 250μL 250μL 250μL 250μL 250μL 250μL 250μL 250μL 1000pg/mL R11000μL 8000pg/mL 250μL 4000pg/mL 250μL 250pg/mL 250μL 500pg/mL 250μL 125pg/mL 250μL 2000pg/mL 250μL 62.5pg/mL 250μL 0pg/mLto prepare2000μL Working Streptavidin-HRP Buffer.Wash Buffer-If crystals have formed in the concentrate,warm to room temperature and mix gently until the crystals have completely dissolved.Dilute1:20with double distilled or deionized water before use,for example:Add20mL of Wash Buffer Concentrate to380mL of deionized or distilled water to prepare 400mL of Wash Buffer.Assay ProcedureBring all reagents and samples to room temperature before use. It is recommended that all standards,controls,and samples be assayed in duplicate.1.Prepare all reagents,working standards,and samples asdirected in the previous sections.2.Remove excess microplate strips from the plate frame,return them to the foil pouch containing the desiccant pack, and reseal properly.3.Add wash buffer350μL/well,aspirate each well afterholding40seconds,repeating the process two times fora total of three washes.4.Add100μL Standard/sample Diluent(R1)in a blank well.5.Add100μL different concentration of standard or samplein other wells,Cover with the adhesive strip provided.Incubate for2hours at37℃.record the plate layout ofstandards and sample assay.6.Prepare the Concentrated Biotin Conjugate Antibody(100x)Working Solution15minutes early before use.7.Repeat the aspiration/wash as in step 3.8.Add100μL Working Biotin Conjugate Antibody in each well,cover with new adhesive Sealer provided.Incubate for1hour at37℃.9.Prepare the Streptavidin-HRP Concentrated(100x)WorkingSolution15minutes early before use.10.Repeat the aspiration/wash as in step 3.11.Add100μL Working Streptavidin-HRP in each well,coverwith new adhesive Sealer provided.Incubate for0.5hourat37℃.12.Repeat the aspiration/wash as in step 3.13.During the incubation,turn on the microplate reader towarm up for30minutes before measuring.14.Add100μL TMB Substrate to each well.Incubate for15-20minutes at37℃.Protect from light.15.Add50μL Stop Solution,determine the optical density ofeach well within5minutes,using a Microplate reader setto450nm.If wavelength correction is available,set to570nm or630nm.If wavelength correction is not available, subtract readings at570nm or630nm from the readingsat450nm.This subtraction will correct for opticalimperfections in the plate.Readings made directly at450nm without correction may cause higher value and lessaccurate result.Assay Procedure SummaryPrepare the standard and reagentsWash3times↓Add100ul of standards or test samples to each well Incubate for2hours at37℃,then wash3times↓Add100ul Working Biotin Conjugate AntibodyIncubate for1hour at37℃,then wash3times↓Add100ul Working Streptavidin-HRPIncubate for0.5hour at37℃,then wash3times↓Add100ul Substrate SolutionIncubate for15-20min at37℃under dark condition↓Add50ul Stop Solution↓Detect the optical density within5minutes under450nm.Correction Wavelength set at570nm or630nmCalculation of Results1.Average the duplicate readings for each standard,controland sample,and subtract the average zero standard optical density(O.D.).2.Create a standard curve by reducing the data using computersoftware 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 a log/log graph.The data may be linearized by plotting the log of the IL-1 beta concentrations versus the log of the O.D.on a linear scale,and the best fit line can be determined by regression analysis.3.If samples have been diluted,the concentration read fromthe standard curve must be multiplied by the dilution factor.Typical DataThe standard curves are provided for demonstration only.A standard curve should be generated for each set of IL-1beta assayed.Detection Range62.5-4000pg/mLSensitivityThe minimum detectable dose(MDD)of IL-1beta typically less than13.22pg/mL.The MDD was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.SpecificityThis assay recognizes both recombinant and natural rat IL-1beta. The factors listed below were prepared at50ng/ml and assayed for cross-reactivity.No significant cross-reactivity was observed with the following:Recombinant rat:Recombinant human:IL-10IL-1IFN-γIL-2IL-1αIL-6IL-2IL-4IL-6IL-8TNF-αNote:Limited by current skills and knowledge,it is impossible for us to complete the cross-reactivity detection between IL-1beta and all the analogues,therefore,cross reaction may still exist.PrecisionIntra-plate Precision3samples with low,middle and high level IL-1beta were tested 20times on one plate,respectively.Intra-Assay:CV<10%Inter-plate Precision3samples with low,middle and high level IL-1beta were tested on3different plates,20replicates in each plate.Inter-Assay:CV<15%Intra-Assay Precision Inter-Assay Precision Sample123123 n202020202020 Mean(pg/mL)116522683535116022593542 Standard deviation47.879.4137.975.4162.6219 CV(%) 4.1 3.5 3.9 6.57.2 6.2RecoveryMatrices listed below were spiked with certain level of IL-1beta and the recovery rates were calculated by comparing the measured value to the expected amount of IL-1beta in samples.Sample Average Recovery（%）Range（%）Cell Culture Media(n=5)10495-113 Serum(n=5)9990-108LinearityThe linearity of the kit was assayed by testing samples spiked with appropriate concentration of IL-1beta and their serial dilutions.The results were demonstrated by the percentage of calculated concentration to the expected.Cell Culture Media(n=5)Serum(n=5) //Average of Expected（%）103951:2Range（%）98-10887-102Average of Expected（%）981021:4Range（%）84-11189-114 Average of Expected（%）961001:8Range（%）86-10692-108Average of Expected（%）981061:16Range（%）91-10596-115Trouble ShootingProblem Possible Cause SolutionHigh Background Insufficient washingSufficiently wash plates asrequired.Ensure appropriateduration and number of washes.Ensure appropriate volume of washbuffer in each well.Incorrect incubationprocedureCheck whether the duration andtemperature of incubation are set upas required.Cross-contamination ofsamples and reagentsBe careful of the operations thatcould cause cross-contamination.Use fresh reagents and repeat thetests.No signal or weak signal Incorrect use ofreagentsCheck the concentration anddilution ratio of reagents.Makesure to use reagents in properorder.Incorrect use ofmicroplate readerWarm the reader up before use.Makesure to set up appropriate mainwavelength and correctionwavelength.Insufficient colourreaction timeOptimum duration of colour reactionshould be limited to15-25minutes.Read too late after stopping the colour reaction Read the plate in5minutes after stopping the reaction.Matrix effect ofsamplesUse positive control.Too much signal Contamination of TMBsubstrateCheck if TMB substrate solutionturns e new TMB substratesolution.Plate sealers reusedUse a fresh new sealer in each stepof experiments.Protein concentrationin sample is too highDo pre-test and dilute samples inoptimum dilution ratio.Poor Duplicates Uneven addition ofsamplesCheck the pipette.Periodicallycalibrate the pipette.Impurities andprecipitates insamplesCentrifuge samples before use.Inadequate mixing ofreagentsMix all samples and reagents wellbefore loading.*For research purposes only.Not for therapeutic or diagnostic purposes.。

大鼠Tyzze ELISA试剂盒使用说明书南京森贝伽现货供应，质量保证，价格优惠，试剂盒首选森贝伽。

本试剂仅供研究使用目的：本试剂盒用于检测大鼠血清，血浆及相关液体样本中大鼠Tyzze水平。

实验原理：本试剂盒采用双抗体夹心酶联免疫法（ELISA）测定标本中大鼠Tyzze。

用纯化的大鼠Tyzze 抗体包被微孔板，制成固相抗体，可与样品中Tyzze相结合，经洗涤除去未结合的抗体和其他成分后再与HRP标记的Tyzze抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。

TMB在HRP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。

用酶标仪在450nm波长下测定吸光度（OD值），与CUTOFF值相比较，从而判定标本中大鼠Tyzze的存在与否。

样本处理及要求：1. 血清：室温血液自然凝固10-20分钟，离心20分钟左右（2000-3000转/分）。

仔细收集上清，保存过程中如出现沉淀，应再次离心。

2. 血浆：应根据标本的要求选择EDTA或柠檬酸钠作为抗凝剂，混合10-20分钟后，离心20分钟左右（2000-3000转/分）。

仔细收集上清，保存过程中如有沉淀形成，应该再次离心。

3. 尿液：用无菌管收集，离心20分钟左右（2000-3000转/分）。

仔细收集上清，保存过程中如有沉淀形成，应再次离心。

胸腹水、脑脊液参照实行。

4. 细胞培养上清：检测分泌性的成份时，用无菌管收集。

离心20分钟左右（2000-3000转/分）。

仔细收集上清。

检测细胞内的成份时，用PBS（PH7.2-7.4）稀释细胞悬液，细胞浓度达到100万/ml左右。

通过反复冻融，以使细胞破坏并放出细胞内成份。

离心20分钟左右（2000-3000转/分）。

仔细收集上清。

保存过程中如有沉淀形成，应再次离心。

5. 组织标本：切割标本后，称取重量。

加入一定量的PBS，PH7.4。

用液氮迅速冷冻保存备用。

标本融化后仍然保持2-8℃的温度。

Rat IgG ELISA Kit产品编号 产品名称包装 PI482Rat IgG ELISA Kit96次产品简介：碧云天的Rat IgG ELISA Kit (Rat IgG Enzyme-Linked ImmunoSorbent Assay Kit)，即大鼠总免疫球蛋白G 酶联免疫吸附检测试剂盒，是一种用于特异性地高灵敏地定量检测大鼠血清、血浆或细胞培养上清液中的IgG 的ELISA 试剂盒。

本产品检测灵敏度高，特异性强，重复性好。

多次重复检测结果表明，最小检出量为5.5ng/ml ，本试剂盒采用特异性抗体识别大鼠IgG ，包括IgG1、IgG2a 、IgG2b 和IgG3，检测值为总的IgG 浓度，板内、板间变异系数均小于10%。

免疫球蛋白G (Immunoglobulin G, IgG)是血清中最主要的免疫球蛋白形式，约占血清中免疫球蛋白总含量的75%，主要参与病原体及有毒物质的识别和清除，具有抗病毒、抗菌剂及免疫调节的功能。

IgG 是一种分子量为150kDa 的大分子糖蛋白，由两条轻链和两条重链组成，每条重链和轻链之间通过二硫键相连。

结构上可以分为可变区域和恒定区域，其中可变区域具有抗原识别位点。

IgG 具有4个亚型，即IgG1、IgG2、IgG3和IgG4。

IgG 也是唯一能够穿过胎盘的抗体，在新生儿抗感染中起到重要作用。

本试剂盒采用双抗体夹心ELISA 法(Sandwich ELISA)检测样品中大鼠IgG 的浓度，其原理见图1。

大鼠IgG 特异的单克隆捕获抗体已预包被于酶标板上，当加入标准品或样品时，其中的大鼠IgG 会与捕获抗体结合。

当加入辣根过氧化物酶标记大鼠IgG 抗体后，辣根过氧化物酶标记大鼠IgG 抗体与大鼠IgG 结合，形成夹心的免疫复合物。

最后加入显色剂TMB 溶液，固相捕获的辣根过氧化物酶就会催化无色的显色剂氧化成蓝色物质，在加入终止液后呈黄色。

通过酶标仪检测450nm 处的吸光度值就能实现定量检测。

（本试剂盒仅供体外研究使用，不用于临床诊断！）Elabscience®一氧化氮（NO）比色法测试盒Nitric Oxide (NO) Colorimetric Assay Kit产品货号：E-BC-K035-S产品规格：50 assays(48 samples)/100 assays(96 samples)检测仪器：紫外-可见光分光光度计（550 nm）使用前请仔细阅读说明书。

如果有任何问题，请通过以下方式联系我们：销售部电话************，************技术部电话131****6790具体保质期请见试剂盒外包装标签。

请在保质期内使用试剂盒。

联系时请提供产品批号(见试剂盒标签)，以便我们更高效地为您服务。

用途本试剂盒适用于检测血清、血浆、及动植物组织样本中的NO含量。

检测原理NO在体内或水溶液中极易氧化生成NO2-，与显色剂生成淡红色偶氮化合物（如下图），生成偶氮化合物的浓度与NO的浓度具有线性关系，通过比色可以间接计算NO的浓度。

试剂一和试剂二的作用，为除去样本有色物质的干扰。

本试剂盒检测组织样本时，需测定总蛋白浓度，推荐使用本公司BCA试剂盒(货号E-BC-K318-M)进行测定。

提供试剂和物品说明：试剂严格按上表中的保存条件保存，不同测试盒中的试剂不能混用。

对于体积较少的试剂，使用前请先离心，以免量取不到足够量的试剂。

所需自备物品仪器：紫外-可见光分光光度计（550nm）、涡旋混匀仪、磁力搅拌器、分析天平、微量移液器（1000 μL，200 μL，100 μL，10 μL）、离心机、烧杯（50 mL，25 mL）。

耗材：枪头（1000 μL，200 μL，10 μL）、EP管（5 mL，2 mL）、吸水纸、擦镜纸、磁力搅拌子。

试剂：双蒸水、生理盐水（0.9% NaCl）或PBS（0.01 M，pH 7.4）。

试剂准备①试剂盒中的试剂平衡至室温。

②试剂四工作液：将试剂四加入37.5 mL双蒸水溶解，混匀即可，2-8℃避光保存2个月。