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PD-1 [Biotinylated] : PD-L1 Inhibitor Screening ELISA Assay Pair  pdf  pdf  pdf


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Materials Provided :

Catalog Components 96tests 480tests
A001-214 Human PD-L1 25 μg 125 μg
A002-214 Human PD-1-Biotin 5 μg 25 μg
A003-214 Streptavidin-HRP 5 μg 25 μg
PD1-NA001 Anti-PD-1 Neutralizing Antibody 20 μg 100 μg

pdf  Download Protocol:
Click here to download the protocol

Introduction:

Immune checkpoint pathway is a focal point of today’s cancer research. PD-1 is one of the best characterized checkpoint proteins. The binding between PD-1 and its ligand PD-L1 suppresses T-cell activation and allows cancer cells to escape from body’s immune surveillance. Therefore, the pharmaceutical inhibition of PD-1 or its ligand has been considered a promising strategy by many oncologists.

Reconstitution:

See Certificate of Analysis for details of reconstitution instruction and specific concentration.

Storage:

See Certificate of Analysis for details of storage conditions.

Be sure to store each component at the proper tempe rature upon arrival

Avoid freeze/thaw cycles upon reconstituted.

Stability:

No activity loss was observed after storage at:
Room temperature (RT) for 1 month in lyophilized state.  -20°C for 1 year in lyophilized state.  -80°C for 2 months under sterile conditions after reconstitution.

Application:

This pair is useful for screening for inhibitors of human PD-1 binding to human PD-L1.

Description:

This inhibitor screening ELISA pair is designed to facilitate the identification and characterization of new PD-1 pathway inhibitors. The assay takes advantage of our in house-developed binding of biotinylated human PD-1 to immobilized human PD-L1 in a functional ELISA assay, and employs a simple colorimetric sandwich ELISA platform. Briefly, we provide you with a human PD-1-Biotin protein, a human PD-L1 protein, an anti-PD-1 neutralizing antibody (as method verified Std.), and streptavidin-HRP reagent. Your experiment will include 4 simple steps: a) Coat the plate with human PD-L1. b) Add your molecule of interest to the tests. c) Add Human PD-1-Biotin to bind the coated human PD-L1. d) Add Streptavidin-HRP followed by TMB or other colorimetric HRP substrate. Finally, the ability of your compound to inhibit PD-1 : PD-L1 binding will be determined by comparing OD readings among different experimental groups.

Quality Assurance:

 

Binding of Biotinylated Human PD-1 to Immobilized Human PD-L1 in a Functional ELISA Assay

Immobilized human PD-L1 protein at 2 μg/mL (100 μL/well) can bind human PD-1-Biotin with a linear range of 0.038 - 0.6 μg/mL when detected by Streptavidin-HRP. Background was subtracted from data points before curve fitting.

Inhibition of PD-1-PD-L1 binding by Anti-PD-1 Neutralizing Antibody (Cat. No. PD1-NA001) measured using the PD-1 [Biotinylated] : PD-L1 Inhibitor Screening ELISA Assay Pair (Cat. No. EP-101)

Serial dilutions of anti-PD-1 neutralizing antibody (Cat. No. PD1-NA001) (1:2 serial dilutions, from 10 μg/mL to 0.078 μg/mL (66.667 nM to 0.521 nM) was added into PD-L1 : PD-1-Biotin binding reactions. The assay was performed according to the above described protocol. Background was subtracted from data points prior to log transformation and curve fitting.

 

 

Troubleshooting Guide

In case of a failed experiment, please check the expiration dates and status of the individual reagents first, and make sure that all reagents have been reconstituted and stored as recommended. In addition, please make sure that all equipment are functioning properly.

Below is a list of common problems that can occur during an assay, and some tips that may help solve the problems and improve your results.

For detailed information or for further support, please contact our technical support team at:
TechSupport@acrobiosystems.com


TROUBLESHOOTING GUIDE
Problem Possible Cause Solutions
High background Insufficient washing or blocking
  1. Be sure the blocking step is performed.
  2. Increase number of washes and the volume Wash Buffer used.
  3. Increase Tween-20 concentration to 0.1% in Wash Buffer.
  4. Make sure Streptavidin-HRPis diluted in Blocking Buffer.
Sample solvent contains inhibiting factors
  1. Run a negative control assay with the solvent alone.
  2. Maintain DMSO level at <1%. Increase protein incubation time.
Contamination
  1. Make sure buffers and samples are prepared, used and stored correctly.
The TMB Substrate Working Solution is not fresh
  1. TMB Substrate Working Solution must be used within 15 minutes after preparation.
Colorimetric signal is erratic Inconsistent pipetting or dilution methods
  1. Make sure pipettors are functioning properly and use a multichannel pipettor if possible.
  2. Use master mixes to minimize errors.
  3. Run duplicates for all tests.
TMB Substrate Working Solution is not completely mixed with the reaction solution
  1. Make sure that TMB Substrate Working Solution is adequately mixed with the reaction solution.
Bubbles in the wells
  1. Tap plate gently to disperse bubbles.
Signal is too high
  1. The concentration of the samples should be adjusted to achieve optimal reading.
  2. Decrease colorimetric HRP substrate incubation time.
Signal of positive control is weak or abnormal Human PD-1-Biotin, Human PD-L1 or streptavidin - HRP may have lost activity
  1. Make sure your proteins are aliquoted into single-use aliquots.
  2. Increase the time of reaction or increase the protein concentration may help in case the protein activity is decreased over time.
Errors in instrument settings
  1. Please check instrument setting.
Substrate Stock Solution is outdated; Incubation temperature is incorrect; Incubation time is not sufficient
  1. Make sure the Substrate Stock Solution is working.
  2. Use proper incubation time and temperature.
Pipetting errors
  1. Make sure that the pipette is calibrated and working properly.
Inadequate color development Incomplete removal of residual buffers during previous steps
  1. Wells should appear dry after aspiration.
Problems with conjugate or color reagents
  1. Color should appear immediately after the reagent is added. Make sure no contamination or residual buffers in the wells before you start the color development process.