Jul 16, 2018
Sandwich KIRA-ELISA Protocol
Forked from Sandwich ELISA Protocol
- Umed T Boltaev1,
- Yves Meyer1
- 1Columbia University
Protocol Citation: Umed T Boltaev, Yves Meyer 2018. Sandwich KIRA-ELISA Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rprd5m6
Manuscript citation:
Boltaev, U., Y. Meyer, F. Tolibzoda, T. Jacques, M. Gassaway, Q. Xu, F. Wagner, Y. Zhang, M. Palmer, E. Holson, and D. Sames. 2017. Multiplex quantitative assays indicate a need for reevaluating reported small-molecule TrkB agonists. Sci. Signal. 10. PMID: 28831019 DOI: 10.1126/scisignal.aal1670
Sadick, M. D., a Intintoli, V. Quarmby, a McCoy, E. Canova-Davis, and V. Ling. 1999. Kinase receptor activation (KIRA): a rapid and accurate alternative to end-point bioassays. J. Pharm. Biomed. Anal. 19: 883–91.
Cazorla, M., A. Jouvenceau, C. Rose, J.-P. Guilloux, C. Pilon, A. Dranovsky, and J. Prémont. 2010. Cyclotraxin-B, the First Highly Potent and Selective TrkB Inhibitor, Has Anxiolytic Properties in Mice. PLoS One 5: 17.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 16, 2018
Last Modified: July 16, 2018
Protocol Integer ID: 13777
Abstract
KIRA-ELISA is used for quantitave detection of phosphorylation level of tyrosine kinase receptors. The protocol could be used for many different receptors. In our work, this protocol has been successful for TrkB and FGFR1 receptors. The underlying principle of the assay is capturing the receptor and detecting phosphorylated tyrosine on the intracellular domain of the receptor using pan phospho-tyrosine antibody. Since tyrosine kinase receptor are phosphorylated at multiple tyrosine residues, pan phospho-tyrosine antibody detects any possible antigen on the receptor, resulting in more sensitive assay. Our assay is based on previously published bioassay by M. Sadick et. al. and M. Cazorla et. al.
Guidelines
Solutions and Buffers:
Note: Do not use sodium azide in any buffers or solutions, as sodium azide inactivates the horseradish-peroxidase enzyme.
Phosphate Buffered Saline (PBS):
80.0 g NaCl
14.4 g Na2HPO4
2.4 g KH2PO4
2.0 g KCl
Add ddH2O up to 10 L, pH to 7.2 with HCl
PBS/Tween:
0.5 ml of Tween-20 in 1 L PBS
Blocking Solution:
10% fetal bovine serum or 1% BSA in PBS. Filter before use to remove particulates.
General References:
1. Davies, C. 1994. The Immunoassay Handbook. D. Wild, Ed. Stockton Press, New York.
2. Abrams, J.S. 1995. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Current Protocols in Immunology (J. Coligan, A. Kruisbeek, D. Margulies, E. Shevach, W. Strober, Eds). John Wiley and Sons, New York. Unit 6.20.
3. Sander, B., et al. 1993. J. Immunol. Meth. 166:201.
4. Abrams, J.S., et al. 1992. Immunol. Rev. 127:5.
Cytokine ELISA Troubleshooting Tips
Poor signal-to-noise ratio
• Try Capture Antibody at 1 – 10 µg/ml (generally 2 µg/ml).
• Try Detection Antibody at 0.25 – 2 µg/ml (generally 1 µg/ml).
• Titrate against each other to obtain optimal dilutions.
Low Sensitivity
• Try overnight incubations of standards and samples at 4°C.
Poor Signal
• If using HRP, avoid sodium azide in wash buffers and diluents, as sodium azide inhibits HRP.
• Verify that appropriate antibody pairs were used and the activity of the samples and/or standards.
• Check the activity of enzyme and substrate by coating with Detection Antibody (1 µg/ml), adding biotinylated avidin and revealing with the appropriate substrate. If the enzyme/substrate is active, a strong signal should be observed.
Poor Standard Curve
• Handling Instructions for standards are lot-specific. Refer to product information for proper handling.
• Recombinant protein vials should be quick-spun for maximum recovery.
• BioLegend suggests that cytokines be stored in a concentrated format (>100 ng/ml) and in the presence of a protein carrier.
High Background
• Increase stringency of washing steps by soaking plates for ~1 minute during washes.
• Determine optimum Capture and Detection Antibody dilutions.
• Increase the dilution of Detection Antibody and/or increase the number of washes after Av-HRP incubation.
Materials
STEP MATERIALS
Anti-TrkB AntibodySino BiologicalCatalog #10047-RP02
TrkB Goat anti-Human, Polyclonal, R&D Systems™Thermo Fisher ScientificCatalog #AF397
Anti-rabbit IgG, HRP-linked Antibody Cell Signaling TechnologyCatalog ##7074
TMB One Solution, 100mlPromegaCatalog #G7431
TMB One Solution, 100mlPromegaCatalog #G7431
Anti-TrkB AntibodySino BiologicalCatalog #10047-RP02
TrkB Goat anti-Human, Polyclonal, R&D Systems™Thermo Fisher ScientificCatalog #AF397
Anti-rabbit IgG, HRP-linked Antibody Cell Signaling TechnologyCatalog ##7074
TMB One Solution, 100mlPromegaCatalog #G7431
TMB One Solution, 100mlPromegaCatalog #G7431
Protocol materials
Anti-TrkB AntibodySino BiologicalCatalog #10047-RP02
TrkB Goat anti-Human, Polyclonal, R&D Systems™Thermo Fisher ScientificCatalog #AF397
Anti-TrkB AntibodySino BiologicalCatalog #10047-RP02
Anti-rabbit IgG, HRP-linked Antibody Cell Signaling TechnologyCatalog ##7074
TMB One Solution, 100mlPromegaCatalog #G7431
Anti-rabbit IgG, HRP-linked Antibody Cell Signaling TechnologyCatalog ##7074
TMB One Solution, 100mlPromegaCatalog #G7431
TMB One Solution, 100mlPromegaCatalog #G7431
TrkB Goat anti-Human, Polyclonal, R&D Systems™Thermo Fisher ScientificCatalog #AF397
TMB One Solution, 100mlPromegaCatalog #G7431
Anti-TrkB AntibodySino BiologicalCatalog #10047-RP02
TrkB Goat anti-Human, Polyclonal, R&D Systems™Thermo Fisher ScientificCatalog #AF397
Anti-rabbit IgG, HRP-linked Antibody Cell Signaling TechnologyCatalog ##7074
TMB One Solution, 100mlPromegaCatalog #G7431
TMB One Solution, 100mlPromegaCatalog #G7431
Coat the Plate
Coat the Plate
Dilute unlabeled capture antibody to a final concentration of 0.5 – 8 µg/ml in PBS and transfer 100 µl to each well of a high affinity, protein-binding ELISA plates (e.g., NUNC Immulon 4 HBX).
Prepare 2 plates: 'pY Plate' and 'Total Protein'. Use Sino Ab for pY plate (1/1000 dilution), R&D Ab for total protein plate (1/500 dilution).
100 µL per well
10 mL per plate
Anti-TrkB AntibodySino BiologicalCatalog #10047-RP02
TrkB Goat anti-Human, Polyclonal, R&D Systems™Thermo Fisher ScientificCatalog #AF397
Seal plate to prevent evaporation. Incubate at 4°C overnight, or 2-3h at RT.
Block the Plate
Block the Plate
Bring the plate to room temperature, flick off the capture antibody solution.
Wash with PBS/Tween (5x).
150 µL per well
Add 1% BSA PBS solution.
100 µL per well
Seal plate and incubate at room temperature for ≥ 1 hour.
01:00:00
Wash with PBS/Tween (5x).
Firmly blot plate against clean paper towels.
Add Standards and Samples
Add Standards and Samples
Transfer lysate from experimental plate (where cells were treated).
80 µL per well for pY plate
20 µL per well for total protein (receptor) plate
Seal the plate and incubate at room temperature at 4°C overnight.
Wash with PBS/Tween (5x).
Note
Washes can be effectively accomplished by filling wells with either a squirt bottle, carboy, manifold dispenser, multi-channel pipettor or automatic plate washer. For increased stringency, after each wash, let the plate stand briefly, flick off the buffer, and blot plates on paper towels before refilling.
Note
Perform at least 3 washes.
Add Detection Antibody
Add Detection Antibody
Dilute the detection antibody to 0.25 – 2 µg/ml in Blocking Solution. Add 100 µl of diluted antibody to each well.
Note
For pY plate: prepare 1/2500 dilution (4 ul of Ab stock to 10 ml of 1% BSA PBS/Tween). For primary cultures, reduce the dilution to 1/1250 (8 ul of Ab stock to 10 ml of 1% BSA PBS/Tween).
For total protein (receptor) plate: prepare working dilution of detecting Ab. Capturing Ab and detecting Ab MUST be from different host animals.
SIncubate at room temperature for 1 hour.
01:00:00
Wash with PBS/Tween (5x).
Note
Perform at least 3 washes.
Total protein plate: Add Horseradish Peroxidase Labeled Secondary Antibody
Total protein plate: Add Horseradish Peroxidase Labeled Secondary Antibody
Dilute the Av-HRP conjugate (Cat. No. 405103) or other enzyme conjugate to its pre-determined optimal concentration in Blocking Buffer (usually between 1/500 – 1/2000). Add 100 µl per well.
Anti-rabbit IgG, HRP-linked Antibody Cell Signaling TechnologyCatalog ##7074
Seal the plate and incubate at room temperature for 30 minutes.
00:30:00
Wash with PBS/Tween (5x).
Note
Perform at least 5 washes.
pY Plate Color Development
pY Plate Color Development
Add TMB One solution and incubate for 30 min at RT
TMB One Solution, 100mlPromegaCatalog #G7431
00:30:00 pY plate color development
100 µL per well
Add 1N HCl solution
100 µL per well
Read the optical density (OD) for each well with a microplate reader set to 405 nm.
Total Protein Plate Color Development
Total Protein Plate Color Development
Add TMB One solution and incubate for 5-30 min at RT
TMB One Solution, 100mlPromegaCatalog #G7431
100 µL per well
Add 1N HCl solution
100 µL per well
Read the optical density (OD) for each well with a microplate reader set to 405 nm.