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1Human Angiotensin ¢ò£¨ANG-¢ò£©ELISA KitCatalog No. CSB-E04493h£¨96T£© This immunoassay kit allows for the in vitro quantitative determination of humanANG-¢ò concentrations in serum, plasma. Expiration date six months from the date of manufacture FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.2PRINCIPLE OF THE ASSAYThe microtiter plate provided in this kit has been pre-coated with anantibody specific to ANG-¢ò. Standards or samples are then added to theappropriate microtiter plate wells with a biotin-conjugated polyclonalantibody preparation specific for ANG-¢ò £ánd Avidin conjugated toHorseradish Peroxidase £¨HRP£© is added to each microplate well andincubated. Then a TMB £¨3,3£§,5,5£§ tetramethyl-benzidine£© substrate solutionis added to each well. Only those wells that contain ANG-¢ò,biotin-conjugated antibody £ánd enzyme-conjugated Avidin will exhibit achange in color. The enzyme-substrate reaction is terminated by theaddition of a sulphuric acid solution £ánd the color change is measuredspectrophotometrically at a wavelength of 450 nm ± 2 nm. Theconcentration of ANG-¢ò in the samples is then determined by comparingthe O.D. of the samples to the standard curve.DETECTION RANGE0.62ng/ml-40ng/ml. The standard curve concentrations used for the ELISA’swere40ng/ml, 20ng/ml, 10 ng/ml,5 ng/ml, 2.5 ng/ml, 1.25ng/ml,0.62 ng/ml.SPECIFICITYThis assay recognizes human ANG-¢ò. No significant cross-reactivity orinterference was observed.SENSITIVITYThe minimum detectable dose of human ANG-¢ò is typically less than0.16ng/ml.The sensitivity of this assay, or Lower Limit of Detection £¨LLD£© was definedas the lowest protein concentration that could be differentiated from zero.3MATERIALS PROVIDEDReagent QuantityAssay plate 1Standard 2Sample Diluent 1 x 20 mlBiotin-antibody Diluent 1 x 10 mlHRP-avidin Diluent 1 x 10 mlBiotin-antibody 1 x 120μlHRP-avidin 1 x 120μlWash Buffer1 x 20 ml£¨25×concentrate£©TMB Substrate 1 x 10 mlStop Solution 1 x 10 mlSTORAGE1. Unopened test kits should be stored at 2-8C upon receipt £ánd themicrotiter plate should be kept in a sealed bag. The test kit may be usedthroughout the expiration date of the kit, provided it is stored asprescribed above. Refer to the package label for the expiration date.2. Opened test plate should be stored at 2-8C in the aluminum foil bagwith desiccants to minimize exposure to damp air. The kits will remainstable until the expiring date shown, provided it is stored as prescribedabove.3. A microtiter plate reader with a bandwidth of 10 nm or less £ánd anoptical density range of 0-3 OD or greater at 450nm wavelength isacceptable for use in absorbance measurement.4REAGENT PREPARATIONBring all reagents to room temperature before use.1. Wash Buffer If crystals have formed in the concentrate, warm up toroom temperature £ánd mix gently until the crystals have completelydissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized ordistilled water to prepare 500 ml of Wash Buffer.2. Standard Centrifuge the standard vial at 6000-10000rpm for 30s.Reconstitute the Standard with 1.0 ml of Sample Diluent. Thisreconstitution produces a stock solution of 40 ng/ml. Allow the standardto sit for a minimum of 15 minutes with gentle agitation prior to makingserial dilutions. The undiluted standard serves as the high standard £¨40ng/ml£©. The Sample Diluent serves as the zero standard £¨0 ng/ml£©.Prepare fresh for each assay. Use within 4 hours £ánd discard after use.3. Biotin-antibody Centrifuge the vial before opening. Dilute to theworking concentration using Biotin-antibody Diluent£¨1:100£©,respectively.4. HRP-avidin Centrifuge the vial before opening. Dilute to the workingconcentration using HRP-avidin Diluent£¨1:100£©, respectively.Precaution: The Stop Solution provided with this kit is an acid solution. Weareye, hand, face, £ánd clothing protection when using this material.OTHER SUPPLIES REQUIRED Microplate reader capable of measuring absorbance at 450 nm, withthe correction wavelength set at 540 nm or 570 nm. Pipettes £ánd pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer.5 An incubator which can provide stable incubation conditions up to37°C±0.5°C.SAMPLE COLLECTION AND STORAGE Serum Use a serum separator tube £¨SST£© £ánd allow samples to clotfor 30 minutes before centrifugation for 15 minutes at 1000 g. Removeserum £ánd assay immediately or aliquot £ánd store samples at -20°C.Centrifuge the sample again after thawing before the assay. Avoidrepeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as ananticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes ofcollection. Assay immediately or aliquot £ánd store samples at -20°C.Centrifuge the sample again after thawing before the assay. Avoidrepeated freeze-thaw cycles.Note: Grossly hemolyzed samples are not suitable for use in this assay.ASSAY PROCEDUREBring all reagents £ánd samples to room temperature before use. It isrecommended that all samples, standards, £ánd controls be assayed in duplicate.All the reagents should be added directly to the liquid level in the well. Thepipette should avoid contacting the inner wall of the well.1. Add 100μl of Standard, Blank, or Sample per well. Cover with theadhesive strip. Incubate for 2 hours at 37°C.2. Remove the liquid of each well, don’t wash.3. Add 100μl of Biotin-antibody working solution to each well. Incubatefor 1 hour at 37°C. Biotin-antibody working solution may appearcloudy. Warm up to room temperature £ánd mix gently until solutionappears uniform.64. Aspirate each well £ánd wash, repeating the process three times for atotal of three washes. Wash: Fill each well with Wash Buffer £¨200μl£© andlet it stand for 2 minutes, then remove the liquid by flicking the plateover a sink. The remaining drops are removed by patting the plate on apaper towel. Complete removal of liquid at each step is essential togood performance.5. Add 100μl of HRP-avidin working solution to each well. Cover themicrotiter plate with a new adhesive strip. Incubate for 1 hour at 37°C.6. Repeat the aspiration £ánd wash three times as step 4.7. Add 90μl of TMB Substrate to each well. Incubate for 10-30 minutes at37°C. Keeping the plate away from drafts £ánd other temperaturefluctuations in the dark.8. Add 50μl of Stop Solution to each well when the first four wellscontaining the highest concentration of standards develop obvious bluecolor. If color change does not appear uniform, gently tap the plate toensure thorough mixing.9. Determine the optical density of each well within 30 minutes, using amicroplate reader set to 450 nm.CALCULATION OF RESULTSUsing the professional soft "Curve Exert 1.3" to make a standard curve isrecommended, which can be downloaded from our web.Average the duplicate readings for each standard, control, £ánd sample andsubtract the average zero standard optical density. Create a standard curveby reducing the data using computer software capable of generating a fourparameter logistic £¨4-PL£© curve-fit. As an alternative, construct a standardcurve by plotting the mean absorbance for each standard on the x-axisagainst the concentration on the y-axis £ánd draw a best fit curve through the7points on the graph. The data may be linearized by plotting the log of theANG-¢ò concentrations versus the log of the O.D. £ánd the best fit line canbe determined by regression analysis. This procedure will produce anadequate but less precise fit of the data. If samples have been diluted, theconcentration read from the standard curve must be multiplied by thedilution factor.LIMITATIONS OF THE PROCEDURE The kit should not be used beyond the expiration date on the kit label. Do not mix or substitute reagents with those from other lots or sources. It is important that the Standard Diluent selected for the standard curvebe consistent with the samples being assayed. If samples generate values higher than the highest standard, dilute thesamples with the appropriate Standard Diluent £ánd repeat the assay. Any variation in Standard Diluent, operator, pipetting technique,washing technique, incubation time or temperature, £ánd kit age cancause variation in binding. This assay is designed to eliminate interference by soluble receptors,binding proteins, £ánd other factors present in biological samples. Untilall factors have been tested in the Immunoassay, the possibility ofinterference cannot be excluded.TECHNICAL HINTS Centrifuge vials before opening to collect contents. When mixing or reconstituting protein solutions, always avoid foaming. To avoid cross-contamination, change pipette tips between additions ofeach standard level, between sample additions, £ánd between reagentadditions. Also, use separate reservoirs for each reagent.8 When using an automated plate washer, adding a 30 second soakperiod following the addition of wash buffer, and/or rotating the plate180 degrees between wash steps may improve assay precision. To ensure accurate results, proper adhesion of plate sealers duringincubation steps is necessary. Substrate Solution should remain colorless or light blue until added tothe plate. Keep Substrate Solution protected from light. SubstrateSolution should change from colorless or light blue to gradations ofblue. Stop Solution should be added to the plate in the same order as theSubstrate Solution. The color developed in the wells will turn from blueto yellow upon addition of the Stop Solution. Wells that are green incolor indicate that the Stop Solution has not mixed thoroughly with theSubstrate Solution.