1Human Aβ1-42 ELISA Kit For the quantitative in vitro determination of Aβ1-42concentrations in Human culture supernates, serum, plasma and tissue.TABLE OF CONTENTSContents PageTABLE OF CONTENTS................................................................ 2INTENDED USE ........................................................................... 3PRINCIPLE................................................................................... 3WARNINGS AND PRECAUTIONS............................................... 4MATERIALS PROVIDED WITH THE KIT..................................... 7MATERIALS REQUIRED BUT NOT PROVIDED......................... 7STORAGR CONDITIONS............................................................. 8REAGENT PREPARATION.......................................................... 9SPECIMEN COLLECTION AND PREPARATION........................ 9ASSAY PROCEDURE................................................................ 10CALCULATION OF RESULTS................................................... 12REFERENCES ........................................................................... 133INTENDED USEAn enzyme immunoassay quantitative measurement in cell culture in vitro Human Aβ1-42 supernates, serum, plasma and tissue.PRINCIPLEThe kit assay Human Aβ1-42 level in the sample，use Purified Human Aβ1-42 antibody to coat microtiter plate wells, make solid-phase antibody, then add Aβ1-42 wells, Combined Aβ1-42 antibody which With HRP labeled , become antibody - antigen - enzyme- antibody complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration ofAβ1-42 in the samples is then determined by comparing the O.D. of the samples to the standard curve.4WARNINGS AND PRECAUTIONS This kit is only for scientific research, and shall not be used as a clinical diagnosis of use. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood. The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil pouch and used in the frame provided. Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step. Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir for dispensing a substrate solution that had previously been used for the conjugate solution may turn solution colored. Do not pour reagents back into vials as reagent contamination may occur. Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells. Do not let wells dry during assay; add reagents immediately after completing the rinsing steps. Allow the reagents to reach room temperature (21-26°C) before starting the test. Temperature will affect the absorbance readings of 5the assay. However, values for the patient samples will not be affected. Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may give false results. Handling should be done in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation. Do not use reagents beyond expiry date as shown on the kit labels. All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes and microtiterplate readers. Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of different plates even of the same lot. The kits may have been shipped or stored under different conditions and the binding characteristics of the plates may result slightly different. Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.6 Some reagents contain Proclin, BND and/or MIT as preservatives. In case of contact with eyes or skin, flush immediately with water. TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant volume of water and skin with soap and abundant water. Wash contaminated objects before reusing them. If inhaled, take the person to open air. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard safety guideline or regulation. For information on hazardous substances included in the kit please refer to Material Safety Data Sheets7MATERIALS PROVIDED WITH THE KITInstruction 1Closure Plate Membrane 2Microelisa Stripplate 12well×8strips 2-8℃Standard0.5ml×1 bottle 0 pg/ml0.5ml×1 bottle 300 pg/ml0.5ml×1 bottle 600 pg/ml0.5ml×1 bottle 1200 pg/ml0.5ml×1 bottle 2400 pg/ml 0.5ml×1 bottle 4800 pg/ml2-8℃Sample Billogical 1ml×1 bottle 2-8℃Chromogen Solution A 6ml×1 bottle 2-8℃Chromogen Solution B 6ml×1/ bottle 2-8℃Stop Solution 6ml×1 bottle 2-8℃HRP-Conjugate Reagent 6ml×1 bottle 2-8℃Wash Buffer Concentrate 20ml×1bottle 2-8℃MATERIALS REQUIRED BUT NOTPROVIDED Microplate reader capable of measuring absorbance at 450 nm. Precision pipettes to deliver 2 ml to 1 ml volumes. Adjustable 10ml -100ml pipettes for reagent preparation. Adjustable 10ml -100ml pipettes for reagent preparation.8 100 ml and 1 liter graduated cylinders. Calibrated adjustable precision pipettes, preferably with disposable plastic tips. (A manifold multi-channel pipette is desirable for large assays.) Absorbent paper. 37°C incubator. Distilled or deionized water. Data analysis and graphing software. Graph paper: linear (Cartesian),log-log or semi-log, or log-logit as desired. Tubes to prepare standard or sample dilutions.STORAGR CONDITIONS When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date. Opened reagents must be stored at 2 °C to 8 °C. Microtiter wells must be stored at 2 °C to 8 °C. Once the foil bag has been opened, care should be taken to close it tightly again. Opened kits retain activity for 8 weeks if stored as described above.9REAGENT PREPARATIONBring all reagents to room temperature before useSPECIMEN COLLECTION ANDPREPARATIONSerum-Use a serum separator tube(SST) and allow samples to clot for 30minutes before centrifugation for 15minutes at approximately 1000 xg.Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C.Plasma-Collect plasma using EDTA or heparin as an anticoagulant.Centrifuge samples for 15 minutes at 1000 x g at 2-8°C within 30minutes of collection. Store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles.Cell culture fluid and other biological fluids-Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles10ASSAY PROCEDURE General Remarks All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming. Once the test has been started, all steps should be completed without interruption. Use new disposal plastic pipette tips for each standard, control or sample in order to avoid cross contamination. Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption. As a general rule the enzymatic reaction is linearly proportional to time and temperature. Determine absorption with an ELISA reader at 450 nm against 620 nm as reference. If no reference wavelength is available, read only at 450nm. If the extinction of the highest standard exceeds the measurement range of the photometer, absorption must be 11measured immediately at 405 nm against 620 nm as reference. Assay Procedure1.Add the standard: according to the order on board hole concentration in 50 ul standard product configured.2. Add samples: a blank hole (blank respectively controlled hole without samples, affinity, and standard reagents, the rest of the enzyme each step for the same operation), sample holes. The enzymes standard bag was board to be added sample, and then 40 u l add meat 10 u l Sample billogical. And when the Sample billogical and in a sample of enzyme panels at the bottom of the hole, try not to touch the hole wall, gently shaking blending.3. Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃. 4. Configurate liquid: 25 times of Wash Concentrate diluted 25 times with distilled water and reserve.5. washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat. 6. add enzyme： Add ELISA reagents 50μl to each well, except the blank well.7. incubate：Operation with 3.8． washing：Operation with 5.129. color： add color reagent A 50μl and color reagent B 50μl to each well. Gently mix, incubate for 15 min at 37℃.10. Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction(the blue color change to yellow color Immediately).11. assay：take blank well as zero , measure the optical densit (OD) at 450 nm after Adding Stop Solution and within 15min.CALCULATION OF RESULTS Calculate the average absorbance values for each set of standards, controls and patient samples. Construct a standard curve by plotting the mean absorbance obtained from each standard against its. Concentration with absorbance value on the vertical(Y) axis and concentration on the horizontal (X) axis. Using the mean absorbance value for each sample determine the corresponding concentration from the standard curve. Automated method: The results in the IFU have been calculated automatically using a 4 PL. (4 Parameter Logistics) curve fit. 4 Parameter Logistics is the preferred calculation method. Other data. Reduction functions may give slightly different results.13 The concentration of the samples can be read directly from this standard curve. Samples with. Concentrations higher than that of the highest standard have to be further diluted. For the calculation of. The concentrations this dilution factor has to be taken into account.REFERENCESREF : Cat.-No.: / Kat.-Nr.: / No.- Cat.: / Cat.-No.: / N.º Cat.: / N.–CatLOT : Lot-No.: / Chargen-Bez.: / No. Lot: / Lot-No.: / Lote N.º: / Lotto n.: :No. of Tests: / Kitgröße: / Nb. de Tests: / No. de Determ.: / N.º de Testes: / Quantità dei tests: : Keep away from heat or direct sun light. / Vor Hitze und direkter Sonneneinstrahlung schützen. /Garder à l’abri de la chaleur et de toute exposition lumineuse. / Manténgase alejado del calor o laluz solar directa. / Manter longe do calor ou luz solar directa. / Non esporre ai raggi solari.: Read instructions before use. / Arbeitsanleitung lesen. / Lire la fiche technique avant emploi. /Lea las instrucciones antes de usar. / Ler as instruções antes de usar. / Leggere le istruzioni prima dell’uso.14 : Store at: / Lagern bei: / Stocker à: / Almacene a :/ Armazenar a :/ Conservare a:
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