Table 1

ELISA validation parameters

Mice seraNT antibodyCT antibodyHuman seraNT antibodyCT antibody
%CV 26.318.35 %CV 18.528.9
Spike in 88.6%±9%96%±3.5% Spike in 102%±12.8%89.8%±7.0%
Dilution recovery 122%±64%123%±19% Dilution recovery 149%±18%139%±10%
Antibody epitope 1-131aa728-737aa Antibody epitope 1-131aa700-747aa
Antibody manufacturer
Cell signalling
Antibody manufacturer Millipore,
Bethyl laboratories
  • (1) Percent coefficient of variance (%CV) was calculated for N-terminal (NT) and C-terminal (CT) against mouse and human epitopes, and was calculated for five different serum samples in three identical plates. The mean concentration for a given samples was determined and divided by the SD. Values were then multiplied by 100 to achieve %CV values. (2) Dilution recovery for NT and CT against mouse and human epitopes was calculated for three different serum samples (n=3), each individually diluted 1:1000 (A) or 1:2000 (B), which is within the working dilution range. Dilution recovery was calculated by ((A)/(B*2))*100. (3) Spike in: (a) human samples: For NT/CT antibody, the neat serum samples was diluted 1:5000 and spiked with 3.1 ng/mL. After calculating the protein conc. via standard curve, the neat values were subtracted from the spiked and divided by the expected concentration, which was multiplied by 100 to reach % spiked in recovery; (b) mouse samples spike in for NT antibodies were done in 1:3000 (neat) spiked with 1.5 ng/mL flSirt1 (n=5). Spike in for mouse CT antibodies was done in 1:5000 (neat) spiked with 3.1 ng/mL flSirt1 (n=3). Last two rows in table include epitope recognition and manufacturer information.