Flex - T™ HLA Class I ELISA Protocol
Prepare the substrate solution approximately 10 minutes before use :
10.34ml DI water 1.2ml of Substrate Buffer 10X concentrate 240μl of ABTS stock solution 120μl of Hydrogen peroxide solution .
Vortex to mix well .
The substrate solution should be at room temperature ( 18 - 25°C ) for optimal reproducible results .
Dilute the 5X Coating Buffer to 1X with DI water Prepare Streptavidin solution to coat the plate :
- 24μl of Streptavidin solution
- 11.976 ml 1X Coating Buffer .
Calculate the amount of 1X Dilution Buffer required and prepare the solution by diluting the 10X concentrated buffer 10 times in DI water before use .
The 1X Dilution Buffer can be stored for up to oneweek at 2 - 8°C .
Prepare Wash Buffer ; dilute the 20X wash buffer with DI water , at least 300ml per plate .
Dilute concentrated HRP - conjugated antibody to 0.3μg / ml in 1X Dilution Buffer just before use .
Prepare12ml per plate .
Dilute the Flex - T™ ELISA Positive Control to prepare a 2.7μM solution :
- 5μl of Flex - T™ ELISA Positive Control at 0.2mg / ml
- 2.9μl of 1X Dilution Buffer Coat the wells of the plate with Streptavidin .
Add 100μl of Streptavidin solution to all wells .
Seal theplate and incubate overnight ( 16 - 18 hrs ) at room temperature ( 18 - 25°C ) .
Discard the coating solution and wash the plate 3 times with at least 300μl Wash Buffer per well and blot residual buffer by firmly tapping plate upside down on absorbent paper .
All subsequent washesshould be performed similarly .
To block non - specific binding and reduce background , add 300μl of 1X Dilution Buffer to all wells .
Seal the plate and incubate for 30 minutes at room temperature ( 18 - 25°C ) .
Prepare three dilutions of the HLA control by serial dilution in 1X Dilution Buffer .
Prepare the controls fresh and keep them on melting ice until usage .
To evaluate the outcome of UV - mediated HLA peptide exchange , dilute a small aliquot of the exchange reaction mixture 300 - fold in 1X Dilution Buffer ( refer to the peptide exchange step in Protocol for fluorescent Flex - T™ generation and antigen specific CD8 + T cell staining ) .
Mix thoroughly .
Discard the Dilution Buffer from the plate and blot the residual buffer .
Pipette 100μl of 1X Dilution Buffer ( blank ) , diluted ELISA controls ( H , M , L , positive , negative , and UV only ) , or exchange reaction mixture dilutions , into the appropriate wells ( recommended in duplicate , see plate map ) .
Seal the plates and incubate for 1 hour at 37°C .
Discard the liquid from the wells and wash 3 times with 300μl of wash buffer per well .
Add 100μl of diluted HRP - conjugated antibody .
Seal the plates and incubate for 1 hour at 37°C .
Discard the liquid from the wells and wash 3 times with 300μl of wash buffer per well .
Add 100μl of substrate solution .
Incubate for 8 minutes at room temperature ( 18 - 25°C ) in the dark on a plate shaker at 400 - 500 rpm .
Add 50μl of Stop Solution to all wells and read at 414nm in an ELISA reader within 30 minutes .
View BioLegend website for Representative Data , and Appendix 1 .
