Human hemoglobin elisa kit

Roughly 50 mg of NHP brain tissue was added to 1 mL of RIPA buffer (Boston Bioproducts) with protease and phosphatase inhibitor tablets (Sigma-Aldrich) in a 2 mL Lysing Matrix D tube (MP Biomedicals). The samples were homogenized using an MP Fastprep-24 (MP Biomedicals), and total protein was quantified using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific) and normalized to 1 mg/mL. Tissue samples were diluted either 1:100 (spinal cord), 1:2000 (brain tissue) or 1:10 (CSF) prior to aSyn protein determination. aSyn protein concentrations were measured using the LEGENDMAX Human Alpha Synuclein ELISA Kit (BioLegend) following the manufacturer’s protocol. CSF hemoglobin levels were also analyzed using the human hemoglobin ELISA kit (Bethyl Laboratories). This was done to assess the impact of blood contamination on the aSyn levels detected in CSF due to the high levels of aSyn contained in RBCs. Samples with greater than 1000 ng/mL HgB were excluded from the CSF analysis.

CSF samples were thawed on ice immediately prior to use, and free hemoglobin levels measured using a human hemoglobin ELISA kit, according to manufacturer’s instructions (Cat# E80-136, Bethyl Laboratories). Briefly, 100 µL of capture antibody (1:100 dilution) was added to each well of a 96-well plate and incubated overnight at 4 °C. The plate was then washed and 100 µL of CSF (diluted 1:10 in blocker casein (Thermo Scientific) in Tris-buffered saline) was added to each plate well for 1 h at room temperature. The plate was washed and 100 µL of detection antibody (1:40,000 dilution) added to each well for 1 h at room temperature. After final washes, 3,3',5,5'-tetramethylbenzidine substrate solution was added to each well for 15 min in the dark at room temperature, and the reaction quenched by adding 100 µL of 1 M HCl. The absorbance was measured at 450 nm using a plate reader. All samples were measured in duplicate, and the final free hemoglobin concentration calculated using a standard curve generated using serially diluted pure human hemoglobin calibrator standard. The limit of detection was 6.25 ng/mL. Total protein concentration in 200 µL CSF was measured using the BCA assay (Thermo Scientific; Rockford, IL).