CONCENTRATION ANALYSIS – ELISA vs. SPR

ELISAelisa

  1. Antigen immobilization (standards + unknowns)
    Wash
  2. Blocking
    Wash
  3. Incubation with primary antibody
    Wash
  4. Incubation with secondary antibody
    Wash
  5. Incubate with enzyme-specific substrate
    Wash
  6. Quantify result with optical plate reader
  7. Generate calibration curves
  8. Evaluate unknowns

Disadvantages:

  • Multiple reagents
  • End-point assay
  • Plate-based denaturation
  • Plate-based epitope inaccessibility
  • Weak affinity antibodies are washed away

spr_chart01

SPR

  1. Antibody immobilization
  2. Flow standard + unknown antigen using 4×1 mode
  3. BiOptix concentration analysis software processes the data (see above)

Advantages:

  • No washing
  • No secondary reagents
  • Real-time data
  • Set up and walk away!

Throughput:

  • In one overnight run, one can process
    110 samples (or 55 in duplicate) and
    8 replicates of a 7 point calibration curve

ANTIBODY SCREENING – ELISA vs. SPR

elisa_vs_sprELISA

  1. Antigen immobilization (standards + unknowns)
    Wash
  2. Blocking
    Wash
  3. Incubation with screening antibodies
    Wash
  4. Incubation with secondary antibody
    Wash
  5. Incubate with enzyme-specific substrate
    Wash
  6. Quantify result with optical plate reader

Time Required:

  • ~5 to 30 hours

Disadvantages:

  • Time consuming
  • End-point assay
  • Plate-based denaturation
  • Plate-based epitope inaccessibility
  • Weak affinity antibodies are washed away

spr_chart02

SPR

  1. Antibody capture
  2. Flow antigen
  3. Regenerate surface
  4. Analyze with easy-to-use, intuitive software (see above)

Time Required:

  • 30 mins per 2 mAbs

Advantages:

  • No washing – set up and walk away
  • Real-time data
  • No long wait steps
  • Get kinetics and affinity as well!

Throughput:

  • Screen 96 samples in one day