TITLE: Flex-T™ HLA Class I ELISA Protocol
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The HLA class I ELISA is an enzyme immunoassay based on the detection of β2-microglobulin subunit of HLA class I complexes, after capturing the complex through the conjugated biotin. To this end, biotinylated HLA class I complex is first captured in streptavidin coated microtiter wells. Subsequently, HRP-conjugated anti-human β2- microglobulin is added to detect intact HLA class I complexes. Only intact HLA class I complexes are recognized. Peptides with high affinity binding will be clearly detected by this ELISA technique, while peptides with a moderate to low binding affinity for HLA class I provide a moderate to non-detectable signal. This protocol is designed to evaluate the efficiency of peptide exchange when using the Flex-T™ system.</div></div>

[STEPS]
?. 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 one week 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. Prepare 12ml per plate.
?. Prepare the substrate solution approximately 10 minutes before use:10.34ml DI water1.2ml of Substrate Buffer 10X concentrate240µl of ABTS stock solution120µl of Hydrogen peroxide solutionVortex to mix wellThe substrate solution should be at room temperature (18-25°C) for optimal reproducible results.
?. Dilute the Flex-T™ ELISA Positive Control to prepare a 2.7µM solution:5µl of Flex-T™ ELISA Positive Control at 0.2mg/ml2.9µl of 1X Dilution Buffer
?. [Assay Procedure]
Coat the wells of the plate with Streptavidin. Add 100µl of Streptavidin solution to all wells. Seal the plate and incubate overnight (16-18 hrs) at room temperature (18-25°C).
?. [Assay Procedure]
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 washes should be performed similarly.
?. [Assay Procedure]
To block non-specific binding and reduce background, add 300µl of 1X Dilution Buffer to all wells.
?. [Assay Procedure]
Seal the plate and incubate for 30 minutes at room temperature (18-25°C).
?. [Assay Procedure]
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.
?. [Assay Procedure]
To evaluate the outcome of UV-mediated HLA peptide exchange, dilute a small aliquot of the exchange reaction mixture 1400-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.
?. [Assay Procedure]
Discard the Dilution Buffer from the plate and blot the residual buffer.
?. [Assay Procedure]
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).
?. [Assay Procedure]
Seal the plates and incubate for 1 hour at 37°C.
?. [Assay Procedure]
Discard the liquid from the wells and wash 3 times with 300µl of wash buffer per well.
?. [Assay Procedure]
Add 100µl of diluted HRP-conjugated antibody.
?. [Assay Procedure]
Seal the plates and incubate for 1 hour at 37°C.
?. [Assay Procedure]
Discard the liquid from the wells and wash 3 times with 300µl of wash buffer per well.
?. [Assay Procedure]
Add 100µl of substrate solution.
?. [Assay Procedure]
Incubate for 8 minutes at room temperature (18-25°C) in the dark on a plate shaker at 400-500 rpm
?. [Assay Procedure]
Add 50µl of Stop Solution to all wells and read at 414nm in an ELISA reader within 30 minutes.Plate map example for the HLA class I ELISA:Pos: Positive exchange control peptide (See table in appendix 1)Neg: Negative exchange control peptide (See table in appendix 1)UV: UV-illuminated conditional HLA complex in the absence of exchange peptideBlank: 1X Dilution BufferH: HLA control HM: HLA control ML: HLA control LP: peptide of choice