Home

Manual

1. j oud C lone Corp SEA118Hu 96 Tests Enzyme linked Immunosorbent Assay Kit For Cluster Of Differentiation 30 Ligand CD30L Organism Species Homo sapiens Human Instruction manual FOR IN VITRO AND RESEARCH USE ONLY NOT FOR USE IN CLINICAL DIAGNOSTIC PROCEDURES 11th Edition Revised in July 2013 INTENDED USE The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of CD30L in human serum plasma tissue homogenates and other biological fluids REAGENTS AND MATERIALS PROVIDEmaD Pre coated ready to use 96 well strip plate Plate sealer for 96 wells Standard Standard Diluent 1x20mL Detection Reagent A 1x12mL Detection Reagent B 1x12mL Wash Buffer 30 x concentrate MATERIALS REQUIRED BUT NOT SUPPLIED 1 Microplate reader with 450 10nm filter 2 Precision single or multi channel pipettes and disposable tips 3 Eppendorf Tubes for diluting samples 4 Deionized or distilled water 5 Absorbent paper for blotting the microtiter plate 6 Container for Wash Solution STORAGE OF THE KITS 1 For unopened kit All the reagents should be kept according to the labels on vials The Standard Detection Reagent A Detection Reagent B and the 96 well strip plate should be stored at 20 C upon receipt while the others should be at 4 C 2 For opened kit When the kit is opened the remaining reagents still need to be sto
2. acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm 10nm The concentration of CD30L in the samples is then determined by comparing the O D of the samples to the standard curve CALCULATION OF RESULTS Average the duplicate readings for each standard control and samples and subtract the average zero standard optical density Construct a standard curve by plotting the mean O D and concentration for each standard and draw a best fit curve through the points on the graph or create a standard curve on log log graph paper with CD30L concentration on the y axis and absorbance on the x axis Using some plot software for instance curve expert 1 30 is also recommended If samples have been diluted the concentration read from the standard curve must be multiplied by the dilution factor TYPICAL DATA In order to make the calculation easier we plot the O D value of the standard X axis against the known concentration of the standard Y axis although concentration is the independent variable and O D value is the dependent variable However the O D values of the standard curve may vary according to the conditions of assay performance e g operator pipetting technique washing technique or temperature effects plotting log of the data to establish standard curve for each test is recommended Typical standard curve below is provided for reference only 1314 segham i reck r Suite 164 H
3. not indicated in the manual a preliminary experiment to determine the validity of the kit is necessary Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals 1314 segham i reck r Suite THL Henson TX TTIW L44 Toll ire ERR a paloma Email maila choud cloneons Rapport Protoaing fon Wahan Hubei 44RA FHI loll free He SA OAT Hp wan cloed clone com bial maika choud lomecom lou d lone 0 rp 6 Due to the possibility of mismatching between antigen from other origin and antibody used in our kits e g antibody targets conformational epitope rather than linear epitope some native or recombinant proteins from other manufacturers may not be recognized by our products 7 Influenced by the factors including cell viability cell number or sampling time samples from cell culture supernatant may not be detected by the kit 8 Fresh samples without long time storage is recommended for the test Otherwise protein degradation and denaturalization may occur in those samples and finally lead to wrong results ASSAY PROCEDURE 1 Determine wells for diluted standard blank and sample Prepare 7 wells for standard 1 well for blank Add 100uL each of dilutions of standard read Reagent Preparation blank and samples into the appropriate wells Cover with the Plate sealer Incubate for 2 hours at 37 C 2 Remove the liq
4. this assay tissues were rinsed in ice cold PBS 0 01mol L pH 7 0 7 2 to remove excess blood thoroughly and weighed before homogenization Minced the tissues to small pieces and homogenized them in 5 10mL of PBS with a glass homogenizer on ice Micro Tissue Grinders woks too The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze thaw cycles to further break the cell membranes After that the homogenates were centrifugated for 5 minutes at 5000xg Remove the supernate and assay immediately or aliquot and store at lt 20 C Other biological fluids Centrifuge samples for 20 minutes at 1000xg Remove particulates and assay immediately or store samples in aliquot at 20 C or 80 C Avoid repeated freeze thaw cycles Note 1 Samples to be used within 5 days may be stored at 4 C otherwise samples must be stored at 20 C lt 1 month or 80 C lt 2 months to avoid loss of bioactivity and contamination 2 Sample hemolysis will influence the result so hemolytic specimen should not be detected 3 When performing the assay bring samples to room temperature REAGENT PREPARATION 1 Bring all kit components and samples to room temperature 18 25 C before use 2 Standard Reconstitute the Standard with 1 0mL of Standard Diluent kept for 10 minutes at room temperature shake gently not to foam The concentration of the standard in the stock solution is 10 000pg mL Please firstly dilute th
5. with certain level of recombinant CD30L and the recovery rates were calculated by comparing the measured value to the expected amount of CD30L in samples serum n 5 85 99 LINEARITY The linearity of the kit was assayed by testing samples spiked with appropriate concentration of CD30L and their serial dilutions The results were demonstrated by the percentage of calculated concentration to the expected 1314 segham i reck Er Suite THL Henson TX TTIW L44 Toll ire ERR a Ap wee cloe cloe Email maila cloud clone_ons Rapport Pretcaimng fon Wahan Hubei 450 FHI Toll free HAS A ER IbAT Hipi an m cloed clome com Email maika heud lomr com lo lo n e Corp 1 16 serum n 5 83 95 90 103 79 91 97 105 EDTA plasma n 5 82 92 94 101 87 96 92 102 heparin plasma n 5 80 104 85 97 81 94 78 93 PRECISION Intra assay Precision Precision within an assay 3 samples with low middle and high level CD30L were tested 20 times on one plate respectively Inter assay Precision Precision between assays 3 samples with low middle and high level CD30L were tested on 3 different plates 8 replicates in each plate CV SD meanX100 Intra Assay CV lt 10 Inter Assay CV lt 12 STABILITY The stability of ELISA kit is determined by the loss rate of activity The loss rate of this kit is less than 5 within the expiration date under appropri
6. ate storage condition To minimize extra influence on the performance operation procedures and lab conditions especially room temperature air humidity incubator temperature should be strictly controlled It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end ASSAY PROCEDURE SUMMARY 1 Prepare all reagents samples and standards 2 Add 100uL standard or sample to each well Incubate 2 hours at 37 C 3 Aspirate and add 100uL prepared Detection Reagent A Incubate 1 hour at 37 C 4 Aspirate and wash 3 times 5 Add 100uL prepared Detection Reagent B Incubate 30 minutes at 37 C 6 Aspirate and wash 5 times 7 Add 90uL Substrate Solution Incubate 15 25 minutes at 37 C 8 Add 50uL Stop Solution Read at 450nm immediately IMPORTANT NOTE 1 Limited by the current condition and scientific technology we can t completely conduct the comprehensive identification and analysis on the raw material provided by suppliers So there might be some qualitative and technical risks to use the kit 2 The final experimental results will be closely related to validity of the products operation skills of the end users and the experimental environments Please make sure that sufficient samples are available 3 Kits from different batches may be a little different in detection range sensitivity and color developing time Please perform the experiment exactly according to the i
7. controlled 4 Washing The wash procedure is critical Complete removal of liquid at each step is essential for good performance After the last wash remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate Insufficient washing will result in poor precision and false elevated absorbance reading 5 Controlling of reaction time Observe the change of color after adding TMB Substrate e g observation once every 10 minutes if the color is too deep add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading TMB Substrate is easily contaminated Please protect it from light The environment humidity which is less than 60 might have some effects on the final performance therefore a humidifier is recommended to be used at that condition TEST PRINCIPLE The microtiter plate provided in this kit has been pre coated with an antibody specific to CD30L Standards or samples are then added to the appropriate microtiter plate wells with a biotin conjugated antibody specific to CD30L Next Avidin conjugated to Horseradish Peroxidase HRP is added to each microplate well and incubated After TMB substrate solution is added only those wells that contain CD30L biotin conjugated antibody and enzyme conjugated Avidin will exhibit a change in color The enzyme substrate reaction is terminated by the addition of sulphuric
8. ct measurement at 450nm immediately Note 1 Assay preparation Keep appropriate numbers of wells for each experiment and remove extra wells from microplate Rest wells should be resealed and stored at 20 C 2 Samples or reagents addition Please use the freshly prepared Standard Please carefully add samples to wells and mix gently to avoid foaming Do not touch the well wall For each step in the procedure total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes This will ensure equal elapsed time for each pipetting step without interruption Duplication of all standards and specimens although not required is recommended To avoid cross contamination change pipette tips between additions of standards samples and reagents Also use separated reservoirs for each reagent 1314 segham i reck Er Suite THL Houston TX PTO L44 Tall free ERR a Ap wes cloe cloe Email maila choud clone_ons Rapport Pretoaimg fon Wahan Hubei 250 FHC Toll free HAS E EHISAT Hip wan cloed clone com Email maile choud cloor com lou d lone 0 rp 3 Incubation To ensure accurate results proper adhesion of plate sealers during incubation steps is necessary Do not allow wells to sit uncovered for extended periods between incubation steps Once reagents are added to the well strips DO NOT let the strips DRY at any time during the assay Incubation time and temperature must be
9. directly is not permitted 2 Prepare standard within 15 minutes before assay Please do not dissolve the reagents at 37 C directly 3 Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction and avoid foaming and mix gently until the crystals are completely dissolved To minimize imprecision caused by pipetting use small volumes and ensure that pipettors are calibrated It is recommended to suck more than 10uL for once pipetting The reconstituted Standards Detection Reagent A and Detection Reagent B can be used only once If crystals have formed in the Wash Solution concentrate 30x warm to room temperature and mix gently until the crystals are completely dissolved 6 Contaminated water or container for reagent preparation will influence the detection result SAMPLE PREPARATION 1 We are only responsible for the kit itself but not for the samples consumed during the assay The user should calculate the possible amount of the samples used in the whole test Please reserve sufficient samples in advance Please predict the concentration before assaying If values for these are not within the range of the standard curve users must determine the optimal sample dilutions for their particular experiments Serum plasma samples require about a 20 fold dilution A suggested 20 fold dilution is 20uL Sample 380uL PBS Sample should be diluted by 0 01mol L PBS PH 7 0 7 2 If the samples are
10. e stock solution to 5 000pg mL and the diluted standard serves as the highest standard 5 000pg mL Then prepare 7 tubes containing 0 5mL Standard Diluent and use the diluted standard to produce a double dilution series according to the picture shown below Mix each tube thoroughly before the next transfer Set up 7 points of diluted standard such as 5 000pg mL 2 500pg mL 1 250pg mL 625pg mL 312pg mL 156pg mL 78pg mL and the last EP tubes with Standard Diluent is the blank as Opg mL 1314 segham i reck Er Suite 164 Houston TX TTIW L44 Toll ire ERR a Ap wee cloe cloe Email maila choud clone_os Rapport Fromage fon Wahan Hubei 450 FHC Toll free Re SR 0AT Hip wen cloed clone com Email maile cheud cloor com gt Cloud Clone Corp SO0pL SOOuL SO0uL 500uL SOQuL SOOuL MINI NANT stock Standard Tube 1 2 3 4 5 6 T 8 9 pg mL 10 000 5 000 2 500 1 250 625 312 156 78 0 3 Detection Reagent A and Detection Reagent B Briefly spin or centrifuge the stock Detection A and Detection B before use Dilute to the working concentration with Assay Diluent A and B respectively 1 100 4 Wash Solution Dilute 2OmL of Wash Solution concentrate 30x with 580mL of deionized or distilled water to prepare 600mL of Wash Solution 1 5 TMB substrate Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again Note 1 Making serial dilution in the wells
11. enson TX PTO L44 Toll ire ERR 4a Ap wes cloe cloe Email mail choud clomeos Rapport Proatoaimg fon Wahan Hubei 450 FHC Toll free HAS A EH ISAT Hip wen cloed clone com Email maile choeud cloor com lt gt Cloud Clone Corp l ey a a iz par n im ar 7 o 6000 5000 4000 3000 2000 1000 0 0 5 d 1 5 2 2 5 3 Optical Density Typical Standard Curve for CD30L Human ELISA DETECTION RANGE 78 5 000pg mL The standard curve concentrations used for the ELISA s were 5 000pg mL 2 500pg mL 1 250pg mL 625pg mL 312pg mL 156pg mL 78pg mL SENSITIVITY The minimum detectable dose of CD30L is typically less than 34pg 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 It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration SPECIFICITY This assay has high sensitivity and excellent specificity for detection of CD30L No significant cross reactivity or interference between CD30L and analogues was observed Note Limited by current skills and knowledge it is impossible for us to complete the cross reactivity detection between CD30L and all the analogues therefore cross reaction may still exist RECOVERY Matrices listed below were spiked
12. in the assay should be controlled Furthermore a preliminary experiment before assay for each batch is recommended 9 Each kit has been strictly passed Q C test However results from end users might be inconsistent with our in house data due to some unexpected transportation conditions or different lab equipments Intra assay variance among kits from different batches might arise from above factors too 10 Kits from different manufacturers with the same item might produce different results since we haven t compared our products with other manufacturers 11 The instruction manual also suits for the kit of 48T but all reagents of 48T kit are reduced by half PRECAUTION The Stop Solution suggested for use with this kit is an acid solution Wear eye hand face and clothing protection when using this material TROUBLE SHOOTING Curve Precision Low 1314 segham i reck Er Suite THL Houston TX PTO L44 Tall ire ERR 4a Ap wee cloe cloe Email maila choud clone_os Rapport Pretoaimg fone Wahan Hubei 450 FHC Toll free HAS A EH ISAT Hip wan cloed clome com Email maile choud cloar com
13. nstruction attached in kit while electronic ones from our website is only for information 4 Do not mix or substitute reagents from one kit lot to another Use only the reagents supplied by manufacturer 1314 segham i reck r Suite THL Houston TX POR L44 Toll ire ERR a Ap wee cloe cloe Email maila kudas Rapport Preatoaimng fon Wahan Hubei 450 FHC Toll free HAS E EH ISAT Hip wan cloed clone com Email maile cheud cloor com Cloud Clone Corp 5 Protect all reagents from strong light during storage and incubation All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism 6 There may be some foggy substance in the wells when the plate is opened at the first time It will not have any effect on the final assay results Do not remove microtiter plate from the storage bag until needed 7 Wrong operations during the reagents preparation and loading as well as incorrect parameter setting for the plate reader may lead to incorrect results A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0 3 O D or greater at 450 10nm wavelength is acceptable for use in absorbance measurement Please read the instruction carefully and adjust the instrument prior to the experiment 8 Even the same operator might get different results in two separate experiments In order to get better reproducible results the operation of every step
14. red according to the above storage condition Besides please return the unused wells to the foil pouch containing the desiccant pack and reseal along entire edge of zip seal 1314 seghom i reck r Suite 164 Henson TX TTIW L44 ball ire ERR a poala Email maila hudas Rapport Pretoaimg fon Wahan Hubei 45 FHC Toll free HAS A EH ISAT Hip wan emn Email maia ei lo lo n e Corp Note It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit For the expiration date of the kit please refer to the label on the kit box All components are stable until this expiration date SAMPLE COLLECTION AND STORAGE Serum Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4 C before centrifugation for 20 minutes at approximately 1000xg Assay freshly prepared serum immediately or store samples in aliquot at 20 C or 80 C for later use Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA or heparin as an anticoagulant Centrifuge samples for 15 minutes at 1000xg at 2 8 C within 30 minutes of collection Remove plasma and assay immediately or store samples in aliquot at 20 C or 80 C for later use Avoid repeated freeze thaw cycles Tissue homogenates The preparation of tissue homogenates will vary depending upon tissue type For
15. uid of each well don t wash Add 100uL of Detection Reagent A working solution to each well Incubate for 1 hour at 37 C after covering it with the Plate sealer 4 Aspirate the solution and wash with 350uL of 1x Wash Solution to each well using a squirt bottle multi channel pipette manifold dispenser or autowasher and let it sit for 1 2 minutes Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper Totally wash 3 times After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against absorbent paper 5 Add 100uL of Detection Reagent B working solution to each well Incubate for 30 minutes at 37 C after covering it with the Plate sealer 6 Repeat the aspiration wash process for total 5 times as conducted in step 4 Add 90uL of Substrate Solution to each well Cover with a new Plate sealer Incubate for 15 25 minutes at 37 C Don t exceed 30 minutes Protect from light The liquid will turn blue by the addition of Substrate Solution 8 Add 50uL of Stop Solution to each well The liquid will turn yellow by the addition of Stop solution Mix the liquid by tapping the side of the plate If color change does not appear uniform gently tap the plate to ensure thorough mixing 9 Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the surface of the liquid Then run the microplate reader and condu

Manual

Contents

Download Pdf Manuals

Related Search

Related Contents