Mouse serum IL-6 duplicate well CV exceeded 15%; is dilution linearity testing required?

A CV over 15% is more commonly caused by uneven sample addition and plate washing. Check pipette calibration and tip consistency. For serum samples, centrifuge at low speed first and take the supernatant to avoid aspirating fibers or lipids. Do not use a shared dilution tube for replicate wells; use two independent dilution tubes and add samples separately. During plate washing, fill each well completely with wash solution and blot dry with consistent motion to avoid residual liquid between wells. During incubation, apply a plate seal and keep the shaker speed uniform. A good standard curve indicates that there is no major problem with the reagents overall. Focusing on practicing sample addition and plate washing can bring down the CV of replicate wells.

High CV in serum replicates makes it necessary to perform dilution linearity. Take 2-3 representative serum samples and prepare 1:2, 1:4, and 1:8 gradients. The back-calculated concentrations should be linear with the dilution factor; if not, it indicates matrix interference or that low-value dilutions fall in the unstable region of the standard curve. Also perform spike recovery at low and high levels to assess accuracy. A good standard curve only means the standard diluent system is fine; the serum matrix is different, and interference is more likely to show up in sample dilution linearity. It is recommended to run dilution linearity first before discussing technique.

After opening, place the remaining strips together with the desiccant back into the aluminum foil bag, seal tightly with a sealing clip or heat sealer, and store at 4°C. Do not place directly in the refrigerator without sealing. Generally, it is recommended to use within 2-4 weeks; the longer the time, the more prone to moisture absorption. Aliquot and freeze standards and detection antibodies according to the instructions to avoid repeated freeze-thaw cycles. For longer-term use, it is best to confirm opened stability with the manufacturer, or bridge with a standard curve in each subsequent batch of experiments to prevent performance degradation of old strips from affecting results.

Mouse serum may contain complement, heterophilic antibodies, or rheumatoid factor, which can cause well-to-well variability within a single sample. Try the serum diluent recommended by the manufacturer or add blocking agents; if it remains high, switch to a high-sensitivity kit with a different capture/detection antibody pair for comparison. Do not test only 1:2; it is recommended to also test 1:4 and 1:8 dilutions. If the back-calculated concentrations differ greatly, it indicates interference or values below the lower limit of quantification. Dilution linearity is more informative than a single replicate CV and also helps determine whether the kit needs to be replaced.