Human Amyloid Beta (A4) Precursor Protein, APP ELISA Kit

Unopened Kit: Store at 2 - 8oC. Donot use past kit expiration date. Opened/Reconstituted Reagents: Standardor Sample Diluent, Biotin-antibody Diluent, HRP-avidin Diluent, TMBSubstrate, Wash Buffer, TMB StopSolution.The above mentioned reagents should be stored for up to 1 month at 2 - 8oC. Microplate Wells: Return unused wellsto the foil pouch containing the desiccant pack, reseal along entire edge ofzip-seal. May be stored for up to 1 month at 2 - 8oC.

serum, plasma and other biological fluids

Human APP ELISA Kit

NM_000484

104760

DNA binding, acetylcholinereceptor binding, copper ion binding, heparin binding, identical proteinbinding, iron ion binding, metal ion binding peptidase activator activity, peptidase inhibitor activity, serine-type endopeptidase inhibitor activity, zinc ion binding

Anantibody specific for Human APP is coated onto the wells of the microtiterplate. Samples and standards of Human APP are pipetted into the wells forbinding to the coated antibody. After washing procedure to remove unboundcompounds, an enzyme-linked antibody specific for Human APP is added to thewells. Following a wash to remove any unbound antibody-enzyme reagent, a substratesolution is added to the wells and color develops in proportion to the amountof Human APP bound in the initial step. The color development is stopped andthe intensity of the color is measured. The magnitude of the absorbance forthis developed color is proportional to the amount of Human APP.

1.Validated microplate reader. 2.Eppendorf Tubes for dilution for samples and standards. 3.Deionized or distilled water. 4.Validated adjustable micropipettes, single and multi-channel. 5.Automatic microtiter plate washer or manual vacuum aspiration equipment. 6. 37oCincubator.

1. Prepare all reagents, working standards, and samples as directed in the previous sections. Diluteoriginal density Standard as follow: Set up 7 points of diluted standard suchas 4, 000 pg/mL, 2, 000 pg/mL, 1, 000 pg/mL, 500 pg/mL, 250 pg/Ml, 125 pg/mL, and62.5 pg/mL. The last EP tubes with Sample Diluent is the blank as 0 pg/mL. 2. Add 100ul ofStandard, Control or Sample per well. Cover with the Microtiter platesealers. Incubate for 1.5 hours at 37oC. 3. Aspirate eachwell and wash, Wash by filling each well with 1 x Wash Buffer using a squirtbottle, multi-channel pipette, manifold dispenser or autowasher. Completeremoval of liquid at each step is essential to good performance. Repeatingthe process twice for a total of four washes. After the last wash, removeany remaining 1× Wash Buffer by aspirating or decanting. Invert the plate andblot it against clean paper towels. 4. Add 100ul ofBiotin-antibody working solution to each well. Cover with the Microtiterplate sealers. Incubate for 1 hour at 37oC. Biotin-antibody working solutionmay appear cloudy. Warm to room temperature and mix gently until solutionappears uniform. 5. Repeat theAspirate/Wash four times. 6. Add 100ul ofHRP-avidin working solution to each well. Cover the microtiter plate with anew adhesive strip. Incubate for 0.5 hours at 37oC. 7. Repeat theAspirate/Wash four times. 8. Add 100ul TMBSubstrate to each well. Mix gently, protected from light and incubatesat 37C for 10-20 min. 9. Add one drop (100ul) of TMB Stop Solution to each well to stop the color reaction. If colorchange does not appear uniform, gently tap the plate to ensure thoroughmixing. 10. Determine theoptical density of each well within 5 minutes, using a microplate reader setto 450 nm.

Average the duplicate readings for each standard, control, and samples and subtractthe average zero standard optical density. Plot the optical density for thestandards versus the concentration of the standards and draw the best curve.The data can be linearized by using log/log paper and regression analysis maybe applied to the log transformation. To determine the sample concentrationof each sample, first find the absorbance value on the y-axis and extend ahorizontal line to the standard curve. At the point of intersection, extend avertical line to the x-axis and read the corresponding sample concentration.If samples have been diluted, the concentration read from the standard curvemust be multiplied by the dilution factor.