OBJECTIVE
To set up a contactin-1 ELISA to titrate antiCNTN1+ patients’ sera.
MATERIALS
- Nunc MaxiSorp ELISA plates
- HVH1 patient sera
- Recombinant Human CNTN1 protein (Sino Biological, 10383-H08H)
- RAH-HRP secondary antibody, Dako
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4
- PBS-Tween 0.1%
PROCEDURE
- Coat ELISA plate with CNTN1 protein at different dilutions (5ugr/mL-0.04ugr/mL) in carbonate/bicarbonate buffer (pH 9.6) and incubate O/N at 4ºC rocking.
- The day after, wash plate 3xPBS-Tween 0.1% and block with milk 3% in PBS-Tween for 1 hour, 100uL per well)
- Add primary antibody (50uL of HVH1 sera 1/100 in milk 3%) and incubate 1h RT
- Wash 3xPBS-Tween 0.1%
- Add secondary antibody RAH 1/10000 in milk 3% and incubate 1h at RT
- Wash 3xPBS-Tween
- Add TMB solution 10′
- Stop reaction with H2SO4
RESULTS
ELISA worked at every dilution tested. Reactivity is present even at lowest protein dilutions (from 0.5ugr/ml mild reactivity). The blanks have no reactivity at all.
CONCLUSIONS
We’ll set up the CNTN1 ELISA with protein concentration of 1ugr/mL
