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? Human TGF beta 1 IQELISA?? Kit is a modified ELISA assay with high sensitivity qPCR readout for the quantitative measurement of Human TGF beta 1 in serum, plasma, and cell culture supernatants. This assay employs an antibody specific for Human TGF beta 1 coated on a 96-well PCR plate. Standards and samples are pipetted into the wells and TGF beta 1 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
- TGF beta 1 Microplate (Item A)**: 96 well PCR plate coated with anti-Human TGF beta 1
- Wash Buffer I Concentrate (20x) (Item B): 25 ml of 20x concentrated solution
- Standards (Item C): 2 vials of recombinant Human TGF beta 1
- Assay Diluent B (Item E): 15 ml of 5x concentrated buffer
- Detection Affinity Reagent for TGF beta 1 (Item F): 2 vials of a 4x concentrated solution of anti-Human TGF beta 1 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.
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