SOP for LBP ELISA after DSS-induced injury

Concentrations of plasma LPS-binding protein (LBP) were examined via Mouse LBP SimpleStep ELISA according to the manufacturer’s protocol (Abcam, #ab269542).

Plasma samples were prepared at a 1:16,000 dilution.

Standards, antibody cocktail containing detector and capture antibodies, and 1x wash buffer were prepared according to the manufacturer’s protocol.

In the plate, 50µL of standard or sample was added to appropriate wells in duplicate.

50µL of antibody cocktail was added to all wells.

The plate was then incubated at RT for 1 hour on a plate shaker at 400 rpm.

Wells were decanted and washed three times with 150µL 1x wash buffer.

100µL of TMB Development Solution was added to each well and allowed to incubate in the dark for 12 minutes with shaking at 400rpm

100µL of Stop Solution was added to each well and placed on the shaker for 1 minute at 400 rpm to mix.

Absorbance of 450nm light was recorded with a FLUOstar Omega microplate reader (BMG Labtech).

The standard curve was created by plotting the blank-subtracted average LBP standard absorbance against the standard LBP concentrations.

Sample LBP concentrations were calculated by interpolating the blank-subtracted average absorbance values against the standard curve, multiplied by the dilution factor.