TITLE: U Mass - Insulin
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">The insulin enzyme-linked immunosorbent assay (ELISA) is a standard immunoassay for the detection of insulin levels in serum samples.  This experiment uses a 96-well plate coated with anti-insulin antibodies and developed with a secondary anti-insulin antibody linked to horseradish peroxidase. The ELISA is developed with 3,3',5,5'-Tetramethylbenzidine (TMB), a chromogenic substrate and read on an automated plate spectrophotometer at 450 nm.  Insulin ELISA may be processed with normal or ultra-sensitive assay kit depending on the expected range of sample concentrations. Serum insulin levels are affected by pancreatic β-cell function and insulin sensitivity.  Serum insulin levels are elevated in obesity and insulin resistance.</div></div>

[STEPS]
?. The microplate should be equilibrated to room temperature prior to opening the foil pouch.
?. Assign microplate strips for a duplicate measurement for the standards, controls, and samples.  The remaining microplate strips can be stored at 2~8°C in the tightly sealed foil pouch containing the desiccant.
?. Pipette 5 or 25 µL of each standard, control, and sample into respective wells.
?. See Reagent Preparation and Certificate of Analysis for control reconstitution instructions.
?. For a 5 µL sample volume, use standard concentrations of 0.188, 0.5, 1.25, 3.75, and 6.9 ng/mL (C-G) and Control Levels 2 and 3.
?. For a 25 µL sample size, use standard concentrations of  0.025, 0.9, 0.188, 0.5, and 1.25 ng/mL (A-E) and Control Levels 1 and 2.
?. Pipette 75 µL of Working Strength Conjugate (see Reagent Preparation) into each well.
?. Cover microplate with a plate sealer, and incubate for 2 hours at room temperature while shaking at 700~900 rpm using a microplate shaker.
?. Decant the contents of the wells, and wash the microplate 6x with 350 µL of Working Strength Wash Buffer per well (see Reagent Preparation) using a microplate washer.
?. Alternatively, fill the wells with Working Strength Wash Buffer using a wash bottle equipped with a wash nozzle. It is not recommended to use a multichannel pipette.
?. Wash buffer must be dispensed with adequate and equal force in order to properly wash the wells.
?. Between washes, invert the microplate to discard the liquid and firmly tap the inverted microplate on absorbent paper towels.
?. After the final wash, remove any residual Wash Buffer and bubbles from the wells by inverting and firmly tapping the microplate on absorbent paper towels.
?. Pipette 100 µL of TMB Substrate into each well.
?. Cover microplate with a plate sealer and incubate for 30 minutes at room temperature while shaking at 700~900 rpm on a microplate shaker.
?. Pipette 100 µL of Stop Solution into each well, and gently shake the microplate to mix the contents.
?. Remove any bubbles before proceeding with the next step.
?. Place the microplate in a microplate reader capable of reading the absorbance at 450 nm.
?. The microplate should be analyzed immediately after the addition of the Stop Solution and within 30 minutes.