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[DESCRIPTION]
<p>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.</p>

[GUIDELINES]
<p><strong>Reagent Preparation<br /></strong>See Appendix 1 for recipes and recommended reagents. All reagents should be prepared or diluted immediately prior to use. Before use, bring all reagents to room temperature (18-25°C) with the exception of the HRP anti-human β2-microglobulin antibody and the ELISA positive control which have to be kept on melting ice to ensure stability. Sodium azide inactivates HRP, do not use sodium azide-containing solutions, nor add sodium azide to the supplied materials. Centrifuge all vials before use (1 minute 3000xg at 4°C). Do not allow wells to stand uncovered or dry for extended periods between incubation steps.</p>
<p> </p>
<p><strong>Materials</strong><br />- Coating Buffer 5X concentrate (Cat# 421701)<br />- Streptavidin solution at 1 mg/ml (BioLegend Cat# 280302)<br />- Wash Buffer 20X concentrate: PBS with 0.05% (v/v) Tween 20 (BioLegend Cat# 421601)<br />- Dilution Buffer 10X concentrate (1 M NaCl, 0.5M Tris, 1% BSA, 0.2% Tween 20, pH8.0. See Appendix 1)<br />- HRP anti-human β2-microglobulin antibody (Cat# 280303)<br />- Flex-T™ ELISA Positive Control (BioLegend Cat# 280301)<br />- Substrate Buffer, 10X concentrate (0.1 M citric acid monohydrate/tri-Sodium citrate dehydrate, pH 4.0, See Appendix 1)<br />- ABTS substrate solution (50X 40mM solution made from e.g. Sigma Cat#A1888, or equivalent. See Appendix 1)<br />- Hydrogen peroxide solution (e.g. Sigma Cat#H3410, or equivalent. See Appendix 1)<br />- Stop Solution (2% [w/v] oxalic acid dehydrate, e.g. Sigma Cat#247537. See Appendix 1)<br />- Nunc™ MaxiSorp™ ELISA Plates, Uncoated (BioLegend Cat# 423501)<br />- Plate sealers (BioLegend Cat# 423601)<br />- Deionized (DI) water</p>
<p> </p>
<p><strong>Equipment</strong><br />- Incubator (37°C)<br />- ELISA plate shaker<br />- Wash bottle or automated microplate washer<br />- Microtiter plate reader for measuring absorbance at 414nm<br />- Adjustable pipettes to measure volumes ranging from 3μl to 1 ml</p>
<p>- Multichannel pipetting devices</p>
<p> </p>
<p><strong><a href="http://www.biolegend.com/media_assets/support_protocol/Protocol%20for%20HLA%20class%20I%20ELISA%2006202016.pdf" target="_blank">Represenative Data and Appendix 1.</a></strong></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/d4recje.jpg" data-src="https://s3.amazonaws.com/pr-journal/d4qecje.jpg" data-ofn="HLA class 1 ELISA.JPG" /></p>

[STEPS]
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