Human Elastin (ELN) ELISA Kit
Contents
1. 4 3 Pipettes and disposable pipette tips 4 4 An ELISA reader capable of measuring absorbance at 450 nm 4 5 An incubator which can provide stable incubation conditions up to 37 C 0 5 C 1 4 Version 3 5 1 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 5 Precautions 5 1 Limited by current skill and knowledge it is impossible for us to complete the cross reactivity detection between this analyte and all it s analogues therefore cross reaction may still exist in other species or materials 5 2 Influenced by the factors including cell viability cell number and also sampling time samples from cell culture supernatant may not be detected by the kit 5 3 The reagents and the plate of this kit and it s technical design parameters are only matched and designed for optimal performance for the undiluted original samples in this assay and owing to the possibility of mismatching between antigen from other resource and antibody used in this kit e g antibody targets conformational epitope rather than linear epitope some native or recombinant proteins from other manufacturers may not be recognized by this kit so please do not substitute reagents from one kit to another and use only the reagents supplied by manufacturer and moreover we will not responsibility for using this kit or part of this kit to do any other experiments such as western blot immunohistochemistry spike recovery and so on arbitrarily2. 5 4 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 or storage conditions or different ambient temperature lab equipment operation pipetting washing incubation temperature or time and kit age Assay variance among wells or kits might arise from these factors too 5 5 Kits from different manufacturers with the same item might produce different results since different manufacturers can use different antigens or antibodies and production processes 5 6 The Stop Solution suggested for use of this kit is an acid solution so please pay enough attention to safety when use it Serum and plasma should be handled as potentially hazardous and capable of transmitting disease Disposable gloves must be worn during the assay procedure since no known test method can offer complete assurance that products derived from blood will not transmit infectious agents Therefore all blood derivatives should be considered potentially infectious and good laboratory practices should be followed 6 Samples Collection and Storage 6 1 Serum Centrifuge serum for approximately 20 minutes at 1000 x g or 3000 rpm within 30 minutes after collection Collect the supernatants carefully assay immediately or store samples in aliquot at 20 C or 80 C Avoid repeated freeze thaw cycles 6 2 Plasma Collect plasma using EDTA or heparin as an anticoagulant Ce
3. USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
4. or 3000 rpm within 30 minutes after collection Collect the supernatants carefully assay immediately or store samples in aliquot at 20 C or 80 C Avoid repeated freeze thaw cycles 6 7 Feces Collect and fully shaking samples in a certain amount of PBS Usually 10mg tissue to 100ul PBS After that centrifugate homogenates for approximately 20 minutes at 1500xg or 5000 rpm Collect the supernatants carefully assay immediately or store samples at 20 C or 80 C Avoid repeated freeze thaw cycles 6 8 Important Notes 2 4 Version 3 5 1 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 6 8 1 Although we have listed most of possible samples but it does NOT mean the analyte exists in all of these listed samples because some analytes only exist in some specific organelles cells or tissues 6 8 2 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 make sure that sufficient samples are available 6 8 3 Fresh samples without long time storage are recommended for assay Otherwise protein degradation and denaturalization may occur in those samples and finally lead to wrong results Samples to be used within 5 days may be stored at 2 8 C otherwise samples must be stored at 20 C lt one month or 80 C lt two months to avoid loss of bioactivity and contamination Avoid repea
5. Human Elastin ELN ELISA Kit Cat No MBS033425 Store All Reagents At 2 C 8 C Package Size 48T Kit or 96T Kit Valid Period Six Months 2 C 8 C IN VITRO RESEARCH USE ONLY NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS 1 Introductions This Quantitative Sandwich ELISA kit is only for in vitro research use only not for drug household therapeutic or diagnostic applications This kit is intended to be used for determination the level of ELN hereafter termed analyte in undiluted original Human body fluids tissue homogenates secretions or feces samples This kit is NOT suitable for assaying non biological sources of substances 2 Performances Sensitivity The sensitivity of this kit is 5 Ong ml Detection Range The detection range of this kit is 25ng ml 800ng ml Specificity No significant cross reactivity or interference between this analyte and analogues is observed Reproducibility Both Intra assay CV and Inter assay CV is less than 15 CV SD mean x100 3 Materials Supplied Items Materials 48 Tests 96 Tests 5 20xWash Solution 15ml 25ml 6 Stop Solution 3 0ml 6 0ml 8 Chromogen Solution B 3 0ml 6 0ml 9 Closure Plate Membrane 2 2 10 Sealed Bags 1 1 f Note The concentration gradients of Standards from Sy to S are followed by 800 400 200 100 50 25ng ml 4 Materials Required but Not Supplied 4 1 Distilled or deionized water 4 2 Absorbent papers or paper towels
6. ause unexpected results or contaminated and moreover the level of this analyte that has been diluted may out of the detection range of the kits 8 8 4 The Sample Diluent are more than PBS it also contains a little stabilizer and preservative It is made well as a Blank Control reagent adjusted zero value for the experiment because the Standards also contain a little stabilizer and preservative so DO NOT use your own PBS or other reagents as a Blank Control reagent even if you have used it to collect your samples 8 8 5 Samples or Reagents Addition Please carefully add samples to wells and mix gently to avoid foaming DO NOT touch the well wall as possible 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 samples although not required is recommended To avoid contamination please use fresh disposable pipette tips for each transfer 8 8 6 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 have been added to the well strips DO NOT let the strips DRY at any time during the assay Incubation time and temperature must be observed 8 8 7 Washing Plate The wash procedure is critical Complete r
7. emoval of liquid at each step is essential to 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 falsely elevated absorbance reading 8 8 8 Controlling of Reaction Time Observe the change of color after adding Substrates e g observation once every 10 minutes Substrates should change from colorless or light blue to gradations of blue The color developed in the wells will turn from blue to yellow after added the Stop Solution If the color turns green it indicate the Stop Solution has not mixed thoroughly 8 8 9 Chromogen Solution B is easily contaminated it should remain colorless or light blue until added to the plate please protect it from light 9 Calculation of Results 9 1 Average the duplicate readings for each standard and sample to subtract average optical density of the Blank Control Vp c 9 2 Using the professional curve fitting software to make a standard curve usually most of the curves are linear and a few curves are quadratic or cubic and calculate the level of this analyte 9 3 Note Any variation in ambient temperature lab equipment operation pipetting washing incubation temperature or time and kit age can cause variation in result Each user should obtain his own standard curve 4 4 Version 3 5 1 FOR RESEARCH USE ONLY NOT FOR
8. ng all reagents and samples to room temperature 18 C 25 C naturally for 30min before starting assay procedures DO NOT use hot water baths to thaw samples or reagents If necessary doing a low speed centrifugation for one or two seconds to concentrate the Standards to the bottom of the vials 8 2 Set Standard wells Sample wells and Blank Control wells add Standard 50ul to each Standard well add Sample SOul to each Sample well add Sample Diluent 50pl to each Blank Control well It is recommended that all Standards samples and Sample Diluent be added in duplicate to the plate 8 3 Add 100u1 of HRP conjugate reagent to each well cover with a Closure Plate Membrane and incubate for 60 minutes at 37 C 8 4 Wash the plate 4 times 8 4 1 Manual Washing Dump the incubation mixtures of the wells into a sink or proper waste container Using pipette or squirt bottle fill each well completely with Wash Solution 1x after about one minute s standing invert and hit the plate onto absorbent papers or paper towels until no moisture appears Repeat this procedure four times Note Hold the sides of the plate frame firmly when washing the plate to assure that all strips remain securely in frame 8 4 2 Automated Washing Aspirate all wells then wash plates four times using Wash Buffer 1x Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350yl well wash After final wash invert plate and blot dry by hit
9. ntrifuge samples for approximately 20 minutes at 1000 x g or 3000 rpm within 30 minutes after collection Collect the supernatants carefully assay immediately or store samples at 20 C or 80 C Avoid repeated freeze thaw cycles 6 3 Blood Collect blood using EDTA or heparin as an anticoagulant Centrifuge samples for approximately 15 minutes at 1500 x g or 5000 rpm within 30 minutes after collection Collect the supernatants carefully assay immediately or store samples in aliquot at 20 C or 80 C Avoid repeated freeze thaw cycles 6 4 Other Body Fluids Lymph Fluid and Cerebrospinal Fluid Centrifuge samples for approximately 20 minutes at 1000 x g or 3000 rpm within 30 minutes after collection Collect the supernatants carefully assay immediately or store samples in aliquot at 20 C or 80 C Avoid repeated freeze thaw cycles 6 5 Tissue homogenates The preparation of tissue homogenates will vary depending upon tissue type Remove excess blood and weighed before homogenization Minced the tissues to small pieces and homogenized them in a certain amount of PBS Usually 10mg tissue to 100u1 PBS After that centrifugate homogenates for approximately 15 minutes at 1500xg or 5000 rpm Collect the supernatants carefully assay immediately or store samples at 20 C or 80 C Avoid repeated freeze thaw cycles 6 6 Secretions Saliva Urine Synovial Fluid and so on Centrifuge samples for approximately 20 minutes at 1000 x g
10. ted freeze thaw cycles 6 8 4 Grossly hemolyzed samples are not suitable for use in this assay so the samples should be centrifugated adequately and no hemolysis or granule was allowed 6 8 5 The kit can not assay the samples which contain sodium azide NaN3 because NaN3 will inhibit the activity of horseradish peroxidase HRP 6 8 6 If the samples are not indicated in the manual a preliminary experiment to determine the validity of this kit is necessary 7 Reagent Preparation and Storage Please store the plate and all reagents at 2 C 8 C 7 1 The valid period of this kit is six months at 2 C 8 C The kit should not be used beyond the expiration date 7 2 Wash Solution 1x Dilute one volume of Wash Solution 20x with nineteen volumes of deionized or distilled water Diluted Wash Solution is stable for one month at 2 C 8 C Undiluted Wash Solution and other reagents are stable for six months at 2 C 8 C 7 3 When the kit is opened please used up all Microelisa Stripplate as soon as possible after removed the plate from the foil pouch The Microelisa Stripplate is detachable so please return the unused wells to the foil pouch containing the desiccant pack and reseal along entire edge of zip seal for preventing damps The remaining reagents still need to be stored at 2 C 8 C 8 Assay Procedures Please do the experiments strictly to follow the assay procedures and DO NOT change any assay procedures arbitrarily 8 1 Bri
11. ting plate onto absorbent paper or paper towels until no moisture appears 8 5 Add Chromogen Solution A 50ul and Chromogen Solution B 50 to each well successively Gently mix and then protect from light to incubate for 15 minutes at 37 C 8 6 Add 50p1 Stop Solution to each well The color in the wells should change from blue to yellow If the color in the wells is green or the color change does not appear uniform gently tap the plate to ensure thorough mixing 8 7 Read the Optical Density O D at 450 nm using an ELISA reader within 15 minutes after adding Stop Solution 3 4 Version 3 5 1 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 8 8 Important Notes 8 8 1 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 8 8 2 Do not remove the plate from the foil pouch until needed 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 8 8 3 The concentration gradients of Standards of this kit have already covered far more than the range of concentration of this analyte in undiluted original samples so please DO NOT use the diluted or non original samples when using our kits please assay the undiluted original samples directly otherwise samples that prepared by chemical lysis buffer may c
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