How many times does the human IL-6 ELISA kit need to dilute serum samples?

After reconstitution, let the standard sit at room temperature for 10-15 minutes, then mix gently by inverting; do not vortex. Perform a 7-point serial dilution according to the instructions, changing the pipette tip at each step, and load samples as soon as possible after dilution, preferably within 15 minutes. For the standard curve, duplicate wells are recommended, and a blank well should be included. When there are many samples, first run a pilot experiment to determine the dilution factor; in the formal experiment, samples should be run at least in duplicate. If you have more than 40 samples to run in duplicate, the standards will occupy 16 wells, and one 96T plate will not be enough, so you need to purchase two plates or reduce sample replicates. It is recommended to prepare everything uniformly with standard/sample diluent to avoid recovery bias caused by directly diluting serum with PBS.

Budget and purpose determination. The initial screening and pre experiment of the project are fully sufficient for domestic large factories, with fast delivery time and convenient after-sales service; The key data for making a big article is that imported literature such as R&D and BioLegend has higher recognition, but the delivery time and price are both high, and single boxes often exceed the budget. Regardless of who is selected, sales are required to provide the quality inspection report for that batch, with a focus on intra/inter batch CV, spiked recovery rate, and natural sample testing data, rather than just advertising. Domestic companies such as Lianke, Xinbosheng, and Dake are more commonly used in the inflammatory cytokine community. Do you have any validation data from serum samples.

First, refer to the "Sample Preparation" section in the manual for human serum IL-6. The linear range varies greatly among different manufacturers. Don't start with a fixed dilution. It is recommended to take 2-3 representative serum samples for the first time and perform three dilutions of 2 times, 5 times, and 10 times. At the same time, run the standard curve to see which dilution falls in the middle of the curve. Healthy individuals typically have low levels of IL-6, which may not be diluted or start at 2-fold; Inflammation or irritation samples may be 5-10 times higher. Please pack and freeze the serum as soon as possible after separation to avoid repeated freezing and thawing. It is recommended to evaluate the matrix effect separately for lipid/hemolytic samples.

With over 40 serum samples, if both samples and standards are run in duplicate wells, one 96T plate definitely won't be enough: 40 samples × 2 = 80 wells, plus the standard curve with 7 points × 2 + blank at approximately 16 wells, totaling exactly 96 wells with no margin, making it easy to run short in actual practice. It is recommended to purchase at least two 96T plates, or one 96T plus one 48T; alternatively, you can initially screen serum samples in single wells, then run positive/suspicious samples in duplicate. If budget is limited, prioritize strip-well 96T plates, which can be resealed if not used up in one go. Don't cut duplicate wells just to save on kits—duplicate wells in ELISA are key to assessing pipetting error.