For measuring IL-6 in mouse tissue homogenates, is any special processing required before ELISA? The background interference is high.

If you do not normalize protein concentration when preparing tissue homogenates, comparability between samples will be very poor. BCA or Bradford quantification is recommended: normalize all samples to the same total protein concentration, for example around 2 mg/mL, then dilute with diluent at 1:2 or 1:5. For the final data, it is recommended to report both IL-6 in pg/mL and pg/mg total protein. If the raw concentration is below the limit of detection, do not rush to draw conclusions; first try a small dilution of 3-fold or 10-fold to see whether it falls within the standard curve. If the CV of replicate wells is large, also check the plate washing: inconsistent blotting dry during manual plate washing and slow pipetting order will both amplify it. Use a multichannel pipette for sample addition, and place standards and samples in different positions on the plate when running replicates.

Directly loading RIPA samples is prone to problems. The ionic detergents and salt concentration in RIPA can affect antigen-antibody binding and may also interfere with the HRP reaction, resulting in high CV between replicate wells and falsely low sample values. It is recommended to first dilute the RIPA supernatant 2-5 fold with the sample diluent provided in the kit before measurement; if interference persists, switch to a mild lysis buffer, such as PBS plus 0.1% Triton X-100 or a commercial tissue lysis buffer plus protease inhibitors. For liver tissue, also ensure thorough centrifugation: centrifuge at 4°C, 12000g or above for 10-15 minutes, collect the clear supernatant, and avoid aspirating lipid. Before changing conditions, perform spike-and-recovery: add the standard to the lysis buffer, calculate the recovery rate; a low recovery indicates matrix interference.

Liver tissue itself contains abundant lipids and proteases; even after homogenization and centrifugation, the supernatant may still contain fine particles or lipid droplets. It is recommended to collect the middle-to-lower clear liquid after centrifugation without touching the upper lipid layer. If necessary, filter with a 0.22 μm filter, but filtration may adsorb some proteins, so spike recovery must be used for confirmation. Protease inhibitors must be freshly added in sufficient amounts, because the tissue homogenization process generates large amounts of proteases. A large CV between replicate wells also requires attention to whether the sample is fully mixed, and the number of freeze-thaw cycles should not be excessive. When data are below the detection limit, do not forcibly assign 0; they can be recorded as below the LLOQ, handled as LLOQ/√2 during statistical analysis, and explained in the methods. This also makes it clear during review or verification.

Matrix mismatch between tissue samples and standards is a common cause of large background differences. Standards are often prepared in buffer or serum matrix, whereas your RIPA supernatant has a complex composition. It is recommended to prioritize diluting samples with the sample diluent or standard diluent provided in the kit, and to use the same diluent system for standards as much as possible, rather than substituting PBS. If the instructions support tissue lysate samples, they usually also provide recommended dilution factors. The fact that your standard curve was fine previously but your samples fell below the detection limit indicates that standard reconstitution was not the issue; the problem lies in sample matrix inhibition. Run two dilutions, such as 1:2 and 1:10. If linearity is poor or recovery is low, interference can basically be judged to be present. In this case, changing the diluent takes priority over changing the kit.