Human serum TNF-α ELISA kit for methodological verification, what if the dilution linearity is poor?

Human serum TNF-α is sensitive to freeze-thaw and matrix status, and repeated freeze-thaw is prone to result drift, which will also cause instability after dilution of high concentration samples. It is recommended to separate the serum in time after blood collection to avoid hemolysis and obvious lipid blood, dispense according to the amount of one test, keep it stable at low temperature, and minimize the number of freeze-thaws. In the methodology, do not fix the dilution factor at the beginning, first select the high value sample for gradient dilution, and select the interval where the recalculation concentration and linear deviation are relatively stable. A standard curve R ² close to 0.98 does not necessarily mean that the sample dilution is linearly qualified, but the calculation deviation, recovery rate and fitting residual of each dilution are also critical.

In the absence of high-speed frozen centrifugation, short-term vortexing and standing cannot completely replace the removal of lipoproteins, cell debris, and fibrin particles, and serum is easy to clog or cause background fluctuations when it is viscous. Conventional low-speed centrifugation can be used first, thoroughly mixed, and then the supernatant can be stagnated and taken. If necessary, the sample dilution or low adsorption filter can be used for treatment according to the instructions. In the process, it is recommended to gently vortex and mix after complete thawing, briefly centrifuge to remove particles, load samples with different dilution multiples for pre-test, set up undiluted and diluted samples on the same plate, and record the sample volume error. If it can only be vortexed, do at least blank, standard and parallelism verification of several samples before deciding whether to use it for formal methodology.