The RayBio® Immuno Qunatitative Enzyme Linked ImumunoSorbent Assay (IQELISA™) is an innovative assay that combines the specificity and ease of use of an ELISA with the sensitivity of real-time PCR. This results in an assay that is simultaneously familiar and cutting edge and enables the use of lower sample volumes while also providing more sensitivity. The RayBio® Human IFN-Gamma IQELISA™ kit is a modified ELISA assay with high sensitivity qPCR readout for the quantitative measurement of human IFN-Gamma in serum, plasma, and cell culture supernatants. This assay employs an antibody specific for human IFN-Gamma coated on a 96-well PCR plate. Standards and samples are pipetted into the wells and IFN-Gamma present in a sample is bound to the wells by the immobilized antibody. The wells are washed and a detection affinity molecule is added to the plates. After washing away unbound detection affinity molecule, primers and a PCR master mix are added to the wells and data is collected using qPCR. Ct values obtained from the qPCR are then used to calculate the amount of antigen contained in each sample, where lower Ct values indicate a higher concentration of antigen.
Standard Curves
Typical Data
Spiking & Recovery Results
Serum spike tests show recovery is 94% with a range of 88 to 102%
Application Notes
Kit Components
IFN-Gamma Microplate (Item A)**: 96 well PCR plate coated with anti-human IFN-Gamma
Wash Buffer I Concentrate (20x) (Item B): 25 ml of 20x concentrated solution
Standards (Item C): 2 vials of recombinant human IFN-Gamma
Assay Diluent B (Item E): 15 ml of 5x concentrated buffer
Detection Affinity Reagent for IFN-Gamma (Item F): 2 vials of a 4x concentrated solution of anti-human IFN-Gamma affinity reagent
IQELISA™ Detection Reagent (Item G): 1 ml of a 10x concentrated stock
Primer Solution (Item I): 1.5 ml vial
PCR Master Mix (Item J): 1.4 ml vial
PCR Preparation buffer (Item K): 1 ml vial of 10x concentrated buffer
Final Wash Buffer (Item L): 10 ml vial of 10x concentrated buffer
**The PCR plate used is a 0.2 ml, non-skirted 96-well plate (ThermoFisher, cat. no.: AB0600). Please ensure compatibility with your PCR machine prior to purchase. For additional information contact technical support (techsupport@raybiotech.com).
Other Materials Required
Real-time PCR instrument, Bio-Rad recommended
Precision pipettes to deliver 2 µl to 1 ml volumes
Adjustable 1-25 ml pipettes for reagent preparation
100 ml and 1 liter graduated cylinders
Absorbent paper
Distilled or deionized water
Log-log graph paper or computer and software for data analysis
Tubes to prepare standard or sample dilutions
Heating block or water bath capable of 80°C
Protocol Outline
Prepare all reagents, samples and standards as instructed
Add 25 µl standard or sample to each well. Incubate for 1.5 - 2.5 hours at room temperature
Add 25 µl detection antibody to each well. Incubate 1 hour at room temperature
Add 15µL Primer solution to each well
Add 10µL of PCR Master Mix to each well
Run real-time PCR
Storage/Stability
The entire kit should be stored for up to 1 year at -20°C from the date of shipment. After use, standard (recombinant protein) should be stored at -20°C or -80°C (recommended at -80°C). Opened PCR plate, Detection Reagent (DNA), Master Mix, and Primers should be stored at -20°C. All other reagents may be stored for up to 1 month at 2° to 8°C. Avoid repeated freeze-thaw cycles.
