Author: Unkown. Date: 12/5/95
Standard Fusion Protein Protocol
Day 1
á Bind Goat anti-mouse Ig (100ug/ml) to ELISA plate in appropriate positions (50ul)
á In separate plate, combine 1 ul FP with appropriate concentration peptide (OVA)
á FP should be diluted in RP10
á Peptide should be diluted in RP10
á Seal and incubate @ 4 C overnight
Day 2
á Remove excess G anti-Mouse and save
á Wash wells 3X with 200 ul RP10
á Add unwashed FP to G anti-Mouse wells @ 50 ul/well
á Seal and incubate @ 4 C overnight OR at room temp for at least 2 hours
á After incubation wash wells 5X with RP10
á Count CTL and add to plate @ 10^5 cells/well (200 ul RP10/well)
á Incubate @ 37 C for 24 hours
Day 3
á Spin down cells
á Harvest 50 ul per well to 2 plates
á Seal and place 2nd plate in -80 C
á Add FDC.P1 cells (4000 cells/well in 50 ul) to 1st plate
á Add controls (ex. 3 wells with RP10 & FDC.P1/3 wells with 45 ul RP10 5 ul GM-CSF & FDC.P1)
á Incubate overnight (At least 8 hours) @ 37 C
Day 4
á Add 10 ul Alamar Blue to wells
á Incubate for 24 hours @ 37 C
Day 5
á Read @ 562 nm, use negative controls for background
BINDING FP DIRECTLY TO PLATE
Day 1
á Add 5 ul FP to elisa plate with 45 ul PBS
á Incubate @ 4 C overnight
Day 2
á Block wells with 150 ul RP10 for 2 hours
á Add peptide in low volume and incubate @ 4 C overnight
Day 3
á Wash plate 4X with 200 ul RP10
á Add CTL and continue Day 2 protocol as above
ETB/1/14/03