ELISA validation parameters
| Mice sera | NT antibody | CT antibody | Human sera | NT antibody | CT antibody |
| %CV | 26.3 | 18.35 | %CV | 18.5 | 28.9 |
| 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 | 1-131aa | 728-737aa | Antibody epitope | 1-131aa | 700-747aa |
|
Antibody manufacturer
(cat#) | Millipore (07–131) | Cell signalling (2028) | Antibody manufacturer | Millipore, (07–131) | Bethyl laboratories (A300-688) |
(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.