ELISA Protocol with Biotinylated Chemokines and CCR7-EMP from Abilita Bio
​Introduction
Biotinylated chemokines have a wide range of assay applications. ​Enzyme-Linked ImmunoSorbent Assay (ELISA) is a powerful technique that can measure the presence of a substance or chemokine with an antibody. Using different streptavidin conjugates, one can study chemokine internalization, receptor binding, or receptor expression levels. Their ability to recognize functional forms of receptors has been exploited by Abilita Bio, our collaborator, to demonstrate the correct folding of their recombinant stabilized receptor (EMP, or Enabled Membrane Protein) in an ELISA assay. 
 
In this protocol, biotinylated CCL19 is used to measure the binding of a purified sample of stabilized CCR7 (CCR7-EMP) generated by Abilita Bio. By using streptavidin-HRP, one can construct the binding curve of CCL19 to CCR7-EMP and calculate the dissociation constant. 
Materials
  • Capture Substance:
    • Solubilized CCR7 receptor - CCR7-EMP [Abilita Bio]
      • Purified to homogeneity
      • Stock concentration: 2 mg/ml in Buffer: 20 mM HEPES pH 7.2, 500 mM NaCl, 0.05 % DDM, 0.01 % CHS
  • Detection Substance:
    • 10 µg CCL19-biotin [ChemoTactics, B-CCL19-10µg]
      • Reconstituted in 9 µl PBS (approximately 100 µM)
  • Negative Control:
    • Control GPCR-EMP [Abilita Bio] 
      • Purified to homogeneity
      • Stock concentration: 1.5 mg/ml; Buffer: 20 mM HEPES pH 7.2, 500 mM NaCl, 0.05 % DDM, 0.01 % CHS
  • Enzyme:
    • Streptavidin-HRP [Pierce, Cat No. 21130]
  • Substrate Solution:
    • Tetramethylbenzidine (TMB) Substrate Solution [Sigma, Cat. No. T0440]
  • Coating Buffer: Phosphate-buffered saline pH 7.4 [Sigma, Cat. No. 79383]
  • Wash Buffer: 20 mM HEPES pH 7.0, 150 mM NaCl, 0.05 % DDM/CHS
  • Binding Buffer: 20 mM HEPES pH 7.2, 150 mM NaCl, 0.05 % DDM/0.01 % CHS + 1% BSA
  • Acquisition Instrument: Molecular Devices SpectraMax i3 plate reader
  • 96-Well Nickel Coated Plates: [Thermo/Pierce, Cat. No. 15442]
Protocol 
  1. Coat each well with 100 µl of CCR7-EMP working solution (0.025 ng/µl, or 2.5 ng in 100 µl of Wash Buffer) at 4°C with rocking, overnight (Figure. A).
  2. Aspirate and wash each well twice with 200 µl Wash Buffer.
  3. Prepare a serial dilution of CCL19-bioitin at 0, 50, 125, 250, 500, and 1,000 nM in 100 µl Binding Buffer. Add to wells and incubate at 4°C for 2 hours (Figure. B)
  4. Aspirate and wash each well twice with 200 µl Wash Buffer.
  5. Prepare a dilution of streptavidin-HRP (1:40,000) in Binding Buffer. Add 100µl to the wells and incubate at 4°C for 15 minutes (Figure. C)
  6. Aspirate and wash each well three times with 200 µl Wash Buffer.
  7. Add 50 µl of TMB solution to each well, develop until blue color is visible and measure the absorbance at 655 nm with a plate reader.
Example Data & Analysis of Results 
Results were analyzed by Prism using one-site binding model to calculate KD = 72 nM.
Picture
Figure D.  ​​Saturation curve measuring B-CCL19 binding to immobilized CCR7-EMP on nickel coated plate. 
Questions about this protocol? Contact us and we can help you. 

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