REAGENTS:
– ELISA plate (nunc immobilizer amino C8)
– PBS
– PBS-tween20 0.05% (PBST)
– BUF033 (biorad)
– BUF037B (biorad)
– TMB (Biolegend)
– Sulphuric acid 25%
– 20F9 (aKIR4.1 – Rat)
– aKIR4.1 – Rabbit (millipore)
– Human sera
– human IgG secondary – HRP (DAKO) (1/3000)
– Rat IgG secondary – HRP (1/3000)
– Rabbit IgG secondary – HRP (DAKO) (1/3000)
ASSAY:
DAY 1
– Dilute protein to different concentration into PBS: 7ug
– Dilute the Flowthrow into PBS: 10ug
– fill each well with 100ul of the protein and PBS and incubate it o/n at 4ºC with shaking.
DAY 2
– Empty the wells and wash it with PBST 3 times
– Add a Blocking buffer (BUF033) 200ul for each well, incubate it 1h at 30ºC.
– Prepare sera after mixing and high centrifugation (13000 rpm). We dilute sera 1/100 in buffer BUF037B. Add 100ul of sera per well and incubate it 30ºC 3 hours on an orbital shaker.
– Wash it with PBST 5 times
– Dilute the appropiate secondary in BUF037B: IgG human 1/3000, IgG rat 1/10000 or IgG rabbit 1/3000. Add 100ul per well and incubate it 30ºC 1h.
– Wash plate 5 times with PBST
– Add 100ul of TMB (dilute 1:1) for 15 minutes. Stop with 50ul of sulphuric acid 25% (6N) [2N equate to 8.34%]
– Read with OD 450nm wavelength
RESULTS:
| S+ | S++ | S+++ | COM | 20F9 | CTRL | |
|---|---|---|---|---|---|---|
| DUP1 | 1.264 | 1.36 | 1.504 | 1.413 | 0.329 | 0.801 |
| DUP1 | 1.304 | 1.286 | 1.584 | 1.41 | 0.321 | 0.854 |
| BLANC1 | 0.452 | 0.576 | 0.519 | 0.061 | 0.063 | 0.562 |
| DUP2 | 1.078 | 1.016 | 1.199 | 0.732 | 0.152 | 0.690 |
| DUP2 | 0.9250 | 1.022 | 1.137 | 0.716 | 0.163 | 0.715 |
| BLANC2 | 0.4269 | 0.576 | 0.467 | 0.06 | 0.061 | 0.448 |
| DUP3 | 0.79 | 0.8509 | 0.860 | 0.992 | 0.127 | 0.661 |
| DUP3 | 0.8090 | 0.8349 | 0.866 | 0.977 | 0.133 | 0.642 |
| BLANC3 | 0.5 | 0.6119 | 0.523 | 0.079 | 0.083 | 0.524 |
CONCLUSIONS:
We observe presence of antibodies in the positive controls, other way we see positivity with millipore’s control. We must improve our background control (FT)
