Streptavidin horseradish peroxidase hrp conjugate

We established a Sandwich ELISA system to identify NUDT15 protein with
high specificity. In brief, 50 μl of the capture Ab (clone #1, 7, 10
μg/ml) in ELISA Coating Buffer (Abcam) was added to each well of a
96-well ELISA plate (Abcam) and the plate was allowed to coat for 1 h at RT. The
plate was washed three times with washing buffer (WB; 1 × PBS containing
0.05% Tween-20) and then blocked with 300 μl of 1 × Blocking
Buffer (Abcam) for 1 h at RT. Then, plates were washed three times with WB and
50 μl samples or standard NUDT15 protein was purified as previously
described⁸. Then, they were added and incubated for 1 h at RT.
Next, plates were washed three times with WB and 50 μl of biotinylated
detection Ab solution (clone #4, 10, 1 μg/ml in 1 × Blocking
Buffer, biotinylated using EZ-Link™ Sulfo-NHS-LC-Biotinylation) was added
and plates were incubated at RT for 15 min. Then plates were washed three times
with WB, and 50 μl of Streptavidin-Horseradish Peroxidase (HRP) Conjugate
(Thermo Fisher Scientific) diluted 1 : 8000 in blocking buffer was added and
incubated at RT for 15 min. The plates were washed three times with WB, and 100
μl of a two-component TMB substrate system (KPL TMB Microwell Peroxidase
Substrate System, SeraCare) was added to each well. After 10 min of incubation
at RT, 100 μl of Stop Solution (Abcam) was added and absorbance was
recorded at 450 nm.

The cells were cultured on 96-well plates (Corning) overnight then transfected with pcDNA-TTR or control plasmid. The culture medium was collected after 24 hours. The medium was analyzed by an Aβ ELISA as follows. Monoclonal 6E10 anti-Aβ(residues 1–17) mouse IgG1, (Biolegend) was coated in 50 mm carbonate buffer, pH 9.6, at 4°C overnight on high binding assay black plates (Costar), washed with TBST (tris buffered saline with 0.05% Tween 20) and blocked with 5% non-fat milk in TBST. Samples and standards (synthetic Aβ_1-40) were incubated for 2 hr, followed by addition of biotin-labeled 4G8 [anti-Aβ residues 17–24, mouse IgG2b (Biolegend)] and incubation for 1 hr at 37°C. After washing, streptavidin horseradish peroxidase (HRP) conjugate (Invitrogen) was added and incubated for 45 min, followed by detection by Quanta Blue fluorogenic peroxidase substrate (Thermo Scientific) using a Tecan Safire II fluorescence plate reader.