Human MICB DuoSet ELISA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Bring all reagents to room temperature before use. Allow all components to sit for a minimum of 15 minutes with gentle agitation after initial reconstitution. -Working dilutions should be prepared and used immediately.	-A standard curve should be generated for each set of samples assayed. -It is suggested to start Reagent Diluent optimization for serum and plasma samples by using PBS supplemented with 10-50% animal serum. -Do not use buffers with animal serum to reconstitute or dilute the Detection Antibody or Streptavidin-HRP B.

Upstream tips
-Bring all reagents to room temperature before use. Allow all components to sit for a minimum of 15 minutes with gentle agitation after initial reconstitution. -Working dilutions should be prepared and used immediately.
Protocol tips
-A standard curve should be generated for each set of samples assayed. -It is suggested to start Reagent Diluent optimization for serum and plasma samples by using PBS supplemented with 10-50% animal serum. -Do not use buffers with animal serum to reconstitute or dilute the Detection Antibody or Streptavidin-HRP B.

Publication protocol

MICA and MICB antibodies in human sera were analyzed with a fluorescence-based sandwich ELISA. Recombinant soluble proteins representing the MICA*008 or MICB*005 extracellular domains were incubated separately at 1 μg/ml in DELFIA® Yellow 96-wells Plates for 12–16 hours at 4 ᵒC. The wells were then washed with TBS / 0.05% Tween-20 and blocked with DELFIA Assay Buffer (PerkinElmer, catalog 1244–111) for 2 hours at room temperature. Human serum samples were diluted 1:1000 in DELFIA Assay Buffer, added to the wells, and incubated for 2 hours at room temperature. The wells were subsequently washed with TBS / 0.05% Tween-20 and incubated for one hour at room temperature with DELFIA Eu-N1 Anti-Human IgG (PerkinElmer, catalog 1244–330) diluted 1:500 in DELFIA Assay Buffer. Wells were then washed with TBS / 0.05% Tween-20 followed by addition of DELFIA Enhancement Solution (PerkinElmer, catalog 1244–105). Time-resolved europium fluorescence was analyzed using an EnVision® Plate Reader (PerkinElmer). Analyses were done in triplicates for each sample.

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Manufacturer protocol

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