Rat Interleukin 10, Il10 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.Theabove 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

Rat Il10 ELISA Kit

NM_012854.2

124092

cytokine activity, interleukin-10, receptor binding, protein binding

Anantibody specific for Rat Il10 is coated onto the wells of the microtiterplate. Samples and standards of Rat Il10 are pipetted into the wells forbinding to the coated antibody. After washing procedure to remove unboundcompounds, an enzyme-linked antibody specific for Rat Il10 is added to thewells. Following a wash to remove any unbound antibody-enzyme reagent, asubstrate solution is added to the wells and color develops in proportion tothe amount of Rat Il10 bound in the initial step. The color development isstopped and the intensity of the color is measured. The magnitude of theabsorbance for this developed color is proportional to the amount of Rat Il10.

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 allreagents, working standards, and samples as directed in the previoussections. Dilute original density Standard as follow: Set up 7 points ofdiluted standard such as 2, 000 pg/mL, 1, 000 pg/mL, 500 pg/mL, 250 pg/mL, 125pg/mL, 62.5 pg/mL and 31.25 pg/mL. The last EP tubes with Sample Diluent isthe 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, remove any remaining 1× Wash Buffer by aspirating or decanting. Invert theplate and blot 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 the Aspirate/Washfour 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.