How many times should serum samples be diluted for mouse TNF-α ELISA kits?

For serum testing, I generally do not recommend directly applying a fixed dilution factor. First, select 2-3 representative animal serum samples and use the diluent provided with the kit to prepare three gradients: 1:2, 1:5, and 1:10. If the OD falls within the middle range of the standard curve, use that dilution; if even 1:2 exceeds the standard curve, continue with 1:20. For splenocyte supernatants, if they have been stimulated, TNF-α may be very high, so you can test at 1:5 or 1:10 first; if unstimulated, dilution is often unnecessary. A pilot experiment must be performed, especially since you only have 20 samples—do not test them all at once and waste them.

With a budget within 1500 and a short lead time required, I commonly use several domestic manufacturers: Xinbosheng, Lianke Bio, Sizhengbo, and Elabscience. A 48T kit is generally sufficient for 20 samples plus the standard curve and replicate wells. Before ordering, confirm that the item is in stock. Do not rely only on the product detail page; ask customer service directly about the shipping time. Usually, in-stock items can arrive in 2-4 days. Also ask whether the kit includes a serum/plasma-matched diluent. Some brands require the diluent to be purchased separately or provide a universal diluent that is not suitable for serum and will increase the CV.

Sensitivity should be at the pg level. It is best to choose a standard curve with the lowest point ≤7.8 pg/mL, or at least below 15 pg/mL, and check whether the sample types clearly stated in the instructions include serum, plasma, and culture supernatant. After arrival, use the standard to run a standard curve, then add replicate wells of serum with a known concentration to verify the CV. Do not directly run precious samples according to the instructions. Regarding quality stability, you can ask the vendor to provide the quality control report for that lot, focusing on inter-assay CV and the lower limit of quantification.

Preliminary experiments should not only determine the dilution factor but also assess dilution linearity. You can perform serial dilution of a high-value serum sample and back-calculate whether the concentrations are consistent; if the back-calculated concentration increases markedly after dilution, it suggests matrix inhibition or interference, and you should increase the dilution or switch to a different sample diluent. For serum samples, also avoid using hemolyzed samples, as hemoglobin can affect color development. For splenocyte supernatants, it is recommended to centrifuge and collect the supernatant to avoid clogging the wells with cell debris.