(MBS810898) PDF
Contents
1. B incubate f 10minutes at 37 C Add stop solution Measure within 15minutes Cat ulation Determining the Results 1 This standard curve is used to determine the contents 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 off the vertical Y axis versus the corresponding concentration on the horizont X axis 2 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 intersection draw a vertical line to the X axis and read the corresponding concentration 3 Any variation in the operator pipetting and washing technique incubation time or temperature and kit age can cause a variation in the result Each user should obtain their own standard curve 5 6 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 3 0 2 0 le 0 5 1 AS standards concentration X this curve is for reference o Package size 96 T gt lt Storage 2 8 C Validity i S PLEASE CAREFULLY READ THIS INSTRUCTION MANUAL BEFORE USE TO BE USED ONLY FOR RESEARCH PURPOSES NOT TO BE USED FOR MEDICAL DIAGNOSIS 6 6 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES2. Mouse total Procollagen Type I Intact N terminal Propeptide total P1NP ELISA Kit instruction MBS810898 Intended use The kit uses a double antibody sandwich enzyme linked immunosorbent one step process assay ELISA to assay the level of total Procollagen Type I Intact N terminal Propeptide total P1 NP in samples Add standard test sample and HRP labeled total Procollagen Type Intact N terminal Propeptide total PINP antibodies to enzyme wells which are Pre coated with total Procollagen Type I Intact N terminal Propeptide total P1NP gantibedy then carry out incubation and wash to remove the uncombined enzyme Upon adding Chromogen Solution A and B the color of the liquid will change into blue and the reaction with the acid will cause the color to become yellow The depth of color and the cofeentration of the total Procollagen Type I Intact N terminal Propeptide total P1 NP sample afe positively correlated Performance Kit 1 Assay range 3 7ng ml 120ng ml 2 Assay method The kit uses a doublecantibody sandwich enzyme linked immunosorbent one step process assay ELISA 3 Characteristics For contentgdetemm ination in serum plasma cell culture supernatant tissue homogenate and any other biological fluid 4 Accuracy Standard lineataeseession correlation coefficient R with the expected value of the concentration great s than or equal to 0 9900 Specificity Thi kitdhas no cross reaction with other soluble structural simila
3. aduated cylinders Absorbent paper Warnings and Precautions l Avoid contact with eyes skin and mucous membrane In case of contact wash affected area with plenty of water immediately Bring kit components out of the fridge for at least 30minutes before its use If the Enzyme coated plates are not used immediately after being opened the remaining plates should be stored in a sealed bag in time The concentrated washing liquid which was removed from the refrigerator may contain some crystals This is a normal phenomenon Please completely dissolve before used Dilute by 1 19 which is 20 times The operation should be carried out in strict accordance with the instructions Test results must be based on the readings of the Enzyme reader For each step add sample by using a pipette which should be calibrated frequently in 2 6 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 10 11 12 13 14 15 order to avoid unnecessary experimental errors In order to avoid cross contamination it is forbidden to re use the tips and seal plate membrane All liquid components should be well mixed before its use Samples should be collected in pyrogen endotoxin free tubes Samples should be frozen if not analyzed shortly after collection Avoid multiple freeze thaw cycles of the samples Thaw completely and mix well prior to analysis When possible avoid use of badly hemolyzed or lipemic serum If large a
4. al fluid Cell culture supernatant When secretory components want to be detected collect with a sterile container Centrifuge for 20 minutes at 2000 3000 rpm collect supernatant When the composition of cells is detected dilute cell suspension with PBS pH 7 2 7 4 work with a cell concentration of 1 million cells ml Centrifuge for 20 minutes at 2000 3000 rpm Collect supernatant If precipitation appears centrifuge again Tissue samples After cutting samples check the weight add PBS PH7 4 Rapidly freeze with liquid nitrogen keep samples at 2 8 C after melting add PBS PAYZ 4 Homogenize by hand or Grinders centrifuge for 20 minutes at the speed of 200043000 rpm Collect supernatant Storage Serum plasma and cell culture fluid samples shouldebeused within 7 days stored at 2 8 C otherwise samples must stored at 20 C 2months or 80 C 6months to avoid loss of bioactivity and contamination Avoid freeze thaw cycles Take out the sample and wait until the temperature turns into rgem temperature before performing the assay DO NOT USE HEAT TREATED SPECIMENS Materials Required but not Supplied SEBO ES ON I S Standard micro plate reader 450nmys Precision pipettes and Disposable pipette tips 37 C incubator Distilled or de ionized wates Data analysis and graphing software Tubes to prepar qstandard or sample dilutions Adjustable L0ml 00mb pipettes for reagent preparation 100 ml and 1 atefgr
5. mounts of particulates are present centrifuge or filter before analysis It is recommended that all standards blank control and samples be run in duplicate The unused reagents shall be put up or covered Do not mix use reagents with different batches and use them before expired date Read absorbances within 2 hours of assay completion Chromogen Solution is light sensitive Avoid prolonged exposure to tight Also avoid contact with metal All samples washing buffer and each kind of waste should be processed according to infective material procedures 1 Standard diluent only used for dilute standard and sed as blank controls 2 Sample diluent only used to dilute sample im the aSsay and there is no other usage Materials Supplied Reagents store at 2 8 C 1x96Wells Pre coated 96 wells 12 8 Strips Ready to use Standard 120ng ml Foi Dilute according to instructions Standard diluent 6 0ml Could be used as blank controls Sample diluent 6 0ml Ready to use HRP Conjugate reagent 6 0ml Ready to use 20X Wash solution 20ml Dilute according to instructions Chromogen Sofutfonj A 6 0ml Ready to use Chromogen Solution B 6 0ml Ready to use Stop Solution 6 0ml Ready to use Microplate Sealers 2 Ready to use User manual 1 Ready to use Sealed bags 1 Ready to use Note Dilute the Standard with Standard diluents in the method of Multiple proportion dilution and the concentrations a
6. r object Repeatability The plate coefficient of variation is less than 15 Plate type Pre Ccoated 12 8 strips 96 wells Reliability Can be performed within two hours Contrary to traditional Elisa methods only a single incubation and wash step is required resulting in fewer handling steps which reduce errors and deliver more consistent results Thorough and regular tests of the system guarantee the stability and reliability of the kit OO ED n Sample Collection and Storage 1 Samples that contain NaN3 cannot be detected because NaN3 inhibits HRP 2 Serum During the operation use non pyrogenic and endotoxin tubes to avoid any cell stimulation collect blood centrifuge 3000 rpm for 10 minutes Carefully separate the serum and red blood cells as quickly as possible If precipitation appears centrifuge again 3 Plasma Use suited EDTA citrate or heparin as an anticoagulant mix 20 minutes centrifuge 30minutes at the speed of 3000 rpm collect supernatant If precipitation appears centrifuge again 1 6 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Homogenate Homogenize with saline buffer and centrifuge for 10minutes at the speed of 3000 rpm then get supernatant for detection Urine Collect with sterile container centrifuge for 20 minutes at 2000 3000 rpm Collect supernatant If precipitation appears centrifuge again Use this description to also process hydrothorax and cerebrospin
7. re as follows 120 60 30 15 7 5 0 ng ml Reagent Preparation 20 x dilution of washing buffer distilled water diluted by 1 20 or 1 copy of the 20 x washing 3 6 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES buffer plus 19 copies of the distilled water Washing Method Manually washing method Empty the plate by inverting it and shaking the content out and tap it on the absorbent papers to dry Add at least 0 35m1 washing solution into each well and soak the plate for 1 2 minutes Repeat this process 5 times Pay attention to avoid spillover Automatic washing method If there is an automatic washing machine it should only be used in the test when you are quite familiar with its function and performance Operation Steps l The quantity of the strips depends on the quantity to be tested samplesand the standards It is suggested to duplicate each standard and blank well Everygsample should be made according to your required quantity and try to use the duplicated wells for samples as well Set blank wells standard wells and test sample wellsg spectively 1 Blank well do not add samples and horseradish ep foxidase HRP other operations are the same 2 Standard wells Add standard 50 to Staridard wells 3 Test sample wells Add 40ul of sample diluent and then add 10ul of sample The final sample dilution is five times and the fal result calculation should be multiplied by fi
8. ve times 4 Add 50ul of horseradish peroxidase HRP into each well except blank well Then seal o the plate and gently shake then in ubate 60 minutes at 37 C Discard Liquid excessfdry ingyfill each well with diluted washing liquid mix and shake for 30 seconds discard the washing liquid and tap the plate into absorbent papers to dry Repeat five times ad then pat dry Add 50ul ochrom gen solution A to each well and then add 50ul of chromogen solution B to each well Gently shake and incubate for 10 minutes at 37 C away from light Stop Add Stop Solution 50ul into each well to stop the reaction the blue changes into yellow immediately Final measurement Set blank well zero measure the optical density OD at 450 nm wavelength which should be carried out within 15 minutes after adding the stop solution According to standards concentration and the corresponding OD values calculate out the standard curve linear regression equation and then apply the OD values of the sample on the regression equation to calculate the corresponding sample s concentration It is acceptable to use a variety of software to make calculations 4 6 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Summary Procedures Prepare reagents samples and standard Add prepared sample and standard and HRP Conjugate Reagent incubate for 60 minutes at 37 C Wash plate five times and add Chromogen Solution A and
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