• Ultra-sensitivity: Up to 1,000x more sensitive than traditional ELISA
• Ultra-low sample volume: Less than 10 µL needed
• Specificity: Sandwich-based detection
• Simplicity: Two steps processing
• Efficiency: Get data within 4 hours
• Multiplex: IL-1 beta, IL-6, IL-12p70, IFN gamma, TNF alpha

*The PCR plate used is a 0.2 mL, non-skirted 96-well plate (Thermo Fisher, cat. no. AB0600). Please ensure compatibility with your PCR machine prior to purchase. For additional information contact technical support (techsupport@raybiotech.com).

• Real-time PCR instrument, Bio-Rad or QuantStudio™ recommended
• Orbital 96-well plate shaker (with ability to reach 700 rpm)
• RayBio^® 96-well Magnetic Plate Separator (Catalog #: BIQ-MPS) or similar
• Optional: Automated microplate washer (BioTek 405 TS Washer recommended) with magnetic separation (RayBio^® 96-well Magnetic Plate Separator recommended)
• Precision pipettes to deliver 1 μL to 1 mL volumes
• Pipette tips (barrier tips are recommended to reduce contamination)
• Adjustable 1-25 mL pipettes for reagent preparation
• Centrifuge that can spin 96 well plates at 500 g
• Nuclease-free water or deionized water
• 1.5 mL, 2 mL, or 5 mL microcentrifuge tubes

1. Prepare all reagents, samples and standards as instructed.
2. Add 5 μL of the bead and conjugate mix, and 5 μL of each standard and sample to each well. Incubate for 2.5 hours at room temperature while shaking.
3. 3. Add 40 µL Assay Diluent and wash the plate using a magnetic plate separator or automated microplate washer.
4. Add 40 μL Elution buffer to each well. Incubate for 10 minutes at room temperature.
5. Prepare PCR/Primer Master Mix and pipette 15 μL into each well of the PCR plate.
6. Transfer 5 μL supernatant from V-bottom plate to the PCR plate.
7. Run real-time PCR.