Objective: To detect protein CNTN1 in blood samples.
Samples:
| CODE | DISEASE |
|---|---|
| 101 | MS |
| 15-198 | CIDP CNTN1+ |
| CTRL - A10A | CTRL - |
| CTRL - M35A | CTRL - |
| Protein CNTN1 | [0.25ug/ml] |
| Protein CNTN1 | [1ug/mL] |
Materials:
- ELISA plates NUNC ref: 442404 Thermoscientific
- Coating buffer: PBS
- Recombinant Human CNTN1 protein
- AF904 abcam anti-CNTN1
- AB66265 Abcam anti-CNTN1
- DAG-peroxidasa Ref. 705-035-147 (box 27)
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4 0.25%
- PBS-Tween20 0.1%
PROCEDURE:
- Coat ELISA plate with Ab66265 (1ugr/mL) in PBS and incubate at RT.
- The day after, wash plate 3xPBS-Tween 0.1% and block with milk 5% in PBS-Tween for 1 hour, 200uL per well).
- Add patients sera diluted 1/100 (200uL/well) and incubate 1h at RT.
- Wash 3xPBS-Tween 0.1%
- Add AF904 (1ugr/ml) in milk 5% and incubate 1h at RT.
- Wash 3xPBS-Tween
- Add DAG-peroxidase diluted 1/5000 in milk 5%
- Wash 3xPBS-Tween
- TMB solution 10′
- Stop reaction with H2SO4
- Lecture at 450-620 nm.
RESULTS:
The ELISA has not worked correctly. We are going to try changing the antibodies and doing it with coating buffer sodium carbonate (pH 9.6) and overnighnt at 4ºc.
