Enzymelinked immunosorbent assay (ELISA)
Blood samples were collected from the tail vein of the rats, centrifuged
at 3000 rpm for 10 min, and serum was collected. The concentration
gradient of the standard was diluted according to the instructions of
the IL-6 ELISA kit and the SP ELISA kit. IL-6 standard concentration
dilution gradient: 160, 80, 40, 20, 10, 5 pg/mL; SP standard
concentration dilution gradient: 8, 4, 2, 1, 0.5, 0.25 ng/mL. 50 μL of
different concentrations of standards were added to each standard well,
and 50 μL of the tested sample was added to the sample well to be
tested. Except for blank wells, 100 μL of horseradish peroxidase
labelled detection antibody was added to each well of standard wells and
sample wells to be tested, gently shaken to mix evenly, and incubated at
37℃ for 1 h. After repeated washing five times, 50 μL of each substrate
A and B were added to each well and incubated at 37 ° C in the dark for
15 min. Finally, 50μL of termination solution was added to each well,
and the liquid changed from blue to yellow to terminate the reaction.
The absorbance values of each well were measured at 450 nm using a
microplate reader.