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Reagent Preparation
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Freshly dilute all reagents and bring all reagents to room temperature before use.
EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent Concentrate 1:10 with reagent grade water. Store for up to 30 days at 2-8°C.
Standard Curve: Reconstitute the 2.4 µg of Human Haptoglobin Standard with 2 mL of EIA Diluent to generate a working solution of 1.2 µg/mL. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the standard solution (1.2 µg/mL) 1:2 with EIA Diluent to generate 0.6, 0.3, 0.15, 0.075, and 0.038 µg/mL solutions. EIA Diluent serves as the zero standard (0 µg/mL). Any remaining solution should be frozen at -20°C and used within 30 days.
Red Fluorescent Human Haptoglobin Probe (1x): Reconstitute the Fluorescent Probe with 1.8 mL EIA Diluent to produce a working solution. Allow the probe to sit for 10 minutes with gentle agitation prior to use. Any remaining solution should be refrigerated at 2-8°C and used within 30 days. Do not freeze.
Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 1:20 with reagent grade water.
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Assay Procedure
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Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is incubated at 37°C.
Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator.
Add 100 µl of Human Haptoglobin Standard or sample per well. Cover wells with a sealing film and incubate for 30 minutes at 37°C. Start the timer after the last addition.
Wash five times with 200 µl of Wash Buffer manually. Invert the plate each time and decant the contents, hit 4-5 times on absorbent material to completely remove the liquid. If using a machine, wash six times with 300 µl of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid.
Add 100 µl of Fluorescent Human Haptoglobin Probe per well. Cover wells with a sealing film and incubate for 30 minutes at 37°C. Start the timer after the last addition.
Wash the microplate as described above.
Immediately add 50 µl of Stabilizing Solution to each well.
Read the endpoint fluorescence on a microplate reader at an excitation wavelength of 488 nm and emission wavelength of 576 nm within 2 hours.
For the Gemini XPS reader, set PMT to High. For the FLx800 reader, a gain of 100 is suggested. For the Synergy H1, a gain of 140 is suggested. However, the user should determine the optional gain/ sensitivity.
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