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Standard curve of human NF-L matched pair antibodies
Sandwich ELISA analysis of human NF-L matched pair antibodiesElisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA723333) diluted in carbonate/bicarbonate buffer, at a concentration of 2ug/ml overnight at 4℃. Wells of the plate were washed, blocked with 150 µl 0.05% tween-20 1% BSA blocking buffer, and incubated with serial diluted Recombinant Human NF-L protein starting from 10,000 pg/ml to 0 pg/ml and detect antibody (HA723334, Biotin, 0.2 µg/ml) for 1 hour at 30℃ with shaking. Then the plate was washed and incubated with 100 µl per well of SA-HRP for 0.5 hour at 30℃ with shaking. Detection was performed using an Ultra TMB Substrate for 10 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.
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Interpolated concentrations of native NF-L in mouse brain, rat brain and U-87 MG extract samples based on a 1000 µg/ml extract load.
Interpolated concentration of native NF-L was measured in duplicate at different sample concentrations and interpolated from the NF-L standard curves. The interpolated dilution factor corrected values were plotted (mean +/- SD, n=2). The mean NF-L concentration was determined to be 934,886 pg/mL in mouse brain and 406.883 pg/mL in rat brain tissue extract. There was no detectable signal in U-87 MG cell extract.
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Interpolated concentrations of spiked NF-L in cell culture media samples.
The concentrations of NF-L were measured in duplicates, interpolated from the NF-L standard curves and corrected for sample dilution. Undiluted samples are as follows: cell culture media 50%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2).
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