Human β amyloid 1 42 elisa kit

We performed sandwich ELISA using Human βAmyloid (1–42) ELISA Kit or Human βAmyloid (1–40) ELISA Kit (298-62401 and 292-62301, FUJIFILM Wako PureChemical Corp., Osaka, Japan). Total proteins extracted from 20 mg of mouse cortex tissues by 1 mL of RIPA buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) were ultra-centrifuged at 100,000 × g at 4 °C for 1 hour. The supernatants were diluted to 100 µL and applied to Aβ1–42 (extract from 100 µg cortex/well) and Aβ1–40 (extract from 500 µg cortex/well) ELISA plates and measured following the manufacturer’s instructions.
The ELISA plates were incubated at 4 °C overnight, washed five times with 1x wash solution, added with 100 µL of HRP-conjugated antibody solution, and incubated at 4 °C for 1 or 2 h in Aβ1–42 or Aβ1–40 ELISA kits, respectively. After washed five times by 1x wash solution, the ELISA plates were added with 100 µL of TMB solution, incubated at room temperature for 30 min, and added with 100 µL of stop solution. Absorbance was measured at 450 nm using a plate reader (SPARK 10 M, TECAN, Grodig, Austria).

We conducted this study using 19 autopsied brain specimens, obtained from 6 AD patients, 7 Cerad-b patients, and 6 normal control subjects who showed no evidence of other CNS disorders in Tokyo Metropolitan Geriatric Medical Center. Frozen postmortem cerebral cortex specimens were used for the measurements. Cerad score is a semiquantitative measure of neuritic plaques and Cerad-b is classified as intermediate probability of AD (Murayama and Saito, 2004 (link)). All the autopsied specimens were obtained from the Brain Bank or Aging Research. The clinical phenotypes, including the diagnostic group (control, Cerad-b, or AD), grades of senile plaque (SPs), and Braak stage were evaluated based on histopathological examination of the specimens. The Ab contents were measured using a Human β Amyloid (1–42) ELISA kit (298–62,401, WAKO Pure Chemical Industries, Osaka, Japan), and adjusted to the brain protein levels. The characteristics of the subjects, as well as ApoE genotypes, are described in Table 1.
The current cross-sectional study was performed in accordance with the ethical guidelines laid down in the Declaration of Helsinki. Written informed consent was obtained in advance from the brain donors and/or the next of kin. The study design was approved by Tokyo Metropolitan Geriatric Medical Center and The University of Tokyo Medical Research Center Ethics Committee (2018088NI).