Q: What is the sensitivity of this kit?
A: This kit can detect between 49 pg/mL and 200,000 pg/mL of 8-isoprostane, which is the range of the standard curve.
Q: What is considered the normal baseline level of 8-isoprotane in humans?
A: The total normal 8-isoprostane levels are about 40-100 pg/mL in human plasma and 500-2000 pg/mL in human urine.
Q: Can the 96-well plate be used over multiple experiments?
A: The plate provided with this kit is a 96-well strip well plate, so any number of strips as necessary can be used and the remained saved for later use. The standard curve will need to repeated each time.
Q: Are EDTA plasma samples compatible with your kits?
A: EDTA will not interfere with this assay and any anticoagulant can be used when preparing plasma samples.
Q: How do I prepare lysates?
A: To prepare cell or tissue lysates for this assay, we recommend resuspending cells or tissues in 1X PBS containing proteinase inhibitors and optional 0.005% Butylated hydroxytoluene, a generic antioxidant available from Sigma-Aldrich; you can prepare 5% stock of BHT in methanol. Homogenize and centrifuge at 12000 xg for 10 min, and harvest the supernatant as the lysate.
Q: What cell number should I use in the ELISA?
A: Unfortunately we do not have a recommended cell number to use when preparing lysates, because this will depend on the level of 8-isoprostane in the sample, which will be different for each researcher. Our recommendation is to start with the most concentrated sample possible and prepare further dilutions later, if necessary, after running a small scale sample titration against the standard curve.
Q: Why is NaOH added to samples?
A: The alkaline hydrolysis is used to free the esterified isoprostane.
Q: Does this assay require a solid phase extraction step?
A: A solid phase extraction step is not required with the sample preparation when using our 8-isoprostane ELISA kit. The main purpose of the solid phase extraction is to release the isoprostane from the lipoprotein complex, which our protocol accomplishes by treating the sample with either acid (urine samples) or base (tissue, serum, and plasma samples). Our kit uses an isoprostane specific antibody that won’t react with other lipids, making it unnecessary to perform the solid phase extraction.
Q: Will the acid interfere with the assay?
A: Low sample pH will interfere with the assay. If the sample pH is not neutral, the antibody and antigen binding during the ELISA will not be optimal, which is why the samples must be diluted at least 1:4 to 1:8 in PBS or Sample Diluent following the acidification step. We recommend checking the pH of samples using a pH strip and further diluting the samples if necessary until the pH is neutral.
Q: How do I know when to add the Stop Solution?
A: A large incubation range is provided because the development time can vary, which is typical for any ELISA. Upon adding the TMB substrate, a blue color will slowly develop, which will then change to yellow after adding the stop solution. If samples are incubated too long in substrate solution they can become saturated. To determine when to add stop solution, focus on the wells with the standard curve where you will want to see a bright blue color develop for the lowest concentration and a very faint color in the highest concentration. When you see a nice gradient of color you should add the stop solution and read the plate immediately. It is important to add the stop solution to all the wells at the same time, using a multichannel pipettor, regardless of the difference in colors between samples.
Q: How do I graph the standard curve?
A: The best way to determine concentrations from an ELISA standard curve is to use a 4-parameter curve fitting program, but if this software is not available it can be done using Excel. When graphing the standard curve the x-axis should be set to logarithmic scale and a linear or logarithmic trendline can be generated. The standard curves generated with ELISAs are not typically linear, but a linear curve can be created by eliminating the upper and lower values of the curve, as long as the sample values fall within this range. The equation of the trendline can be used to calculate the concentrations of unknowns by solving for x in y=mx+b, which is:(OD value-b)/m for linear lines or ln(x)=(OD value-b)/m for logarithmic lines.