ELISA
- Antigen immobilization (standards + unknowns)
Wash - Blocking
Wash - Incubation with primary antibody
Wash - Incubation with secondary antibody
Wash - Incubate with enzyme-specific substrate
Wash - Quantify result with optical plate reader
- Generate calibration curves
- Evaluate unknowns
Disadvantages:
- Multiple reagents
- End-point assay
- Plate-based denaturation
- Plate-based epitope inaccessibility
- Weak affinity antibodies are washed away
SPR
- Antibody immobilization
- Flow standard + unknown antigen using 4×1 mode
- 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
- Antigen immobilization (standards + unknowns)
Wash - Blocking
Wash - Incubation with screening antibodies
Wash - Incubation with secondary antibody
Wash - Incubate with enzyme-specific substrate
Wash - 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
- Antibody capture
- Flow antigen
- Regenerate surface
- 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