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Introduction
The RayBio®Immuno Quantitative Enzyme Linked ImumunoSorbent Assay (IQELISA™) is an innovative new 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® Bovine TNF alpha IQELISA™ Kit is a modified ELISA assay with high sensitivity qPCR readout for the quantitative measurement of Bovine TNF alpha in serum, plasma, and cell culture supernatants. This assay employs an antibody specific for Bovine TNF alpha coated on a 96-well PCR plate. Standards and samples are pipetted into the wells and TNF alpha 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.
Comparison
Standard Curve
Typical Data
Application Notes
Kit Components
TNF alpha Microplate (Item A)**: 96 well PCR plate coated with anti-Bovine TNF alpha
Wash Buffer I Concentrate (20x) (Item B): 25 ml of 20x concentrated solution
Standards (Item C): 2 vials of recombinant Bovine TNF alpha
Assay Diluent B (Item E): 15 ml of 5x concentrated buffer
Detection Affinity Reagent for TNF alpha (Item F): 2 vials of a 4x concentrated solution of anti-Bovine TNF alpha 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 25 µl of IQELISA™ Detection Reagent to each well. Incubate 1 hour
Add 10 µl Primer solution and 10 µl of PCR master mix to each well
Run real-time PCR
Storage/Stability
May be stored for up to 6 months at 2° to 8°C from the date of shipment. Standard (recombinant protein) should be stored at -20°C or -80°C (recommended at -80°C) after reconstitution. Opened PCR plate or reagents may be stored for up to 1 month at 2° to 8°C. Note: the kit can be used within one year if the whole kit is stored at -20°C. Avoid repeated freeze-thaw cycles.
