Table 2 Representative methodologies for experimental affinity validation systems
Methodology | Principle | Advantages | Limitations | Throughput | Kinetic Data? |
|---|---|---|---|---|---|
Enzyme-Linked Immunosorbent Assay (ELISA) | Antigen is immobilized on a solid phase, and antibody binding is detected using an enzyme-linked secondary antibody | Versatile, well-established, relatively low cost | Not as good compared to other methods for quantitation, potential for high background signal | Moderate to High (96-well format common) | No |
Single-Molecule Counting (SMC) | Fluorescently labeled antibodies are used to detect and quantify individual antibody-antigen complexes | High sensitivity (sub-pg/mL), quantitative, allows multiplexing, faster read times than ELISA | Requires specialized equipment, not usually used for antigen–antibody affinity analysis | High (384-well format) | No |
Bio-layer Interferometry (BLI) | Measures changes in interference patterns caused by antibody-antigen binding on a sensor | Label-free real-time analysis, suitable for crude samples | Requires specialized equipment, lower throughput than ELISA | Low to Moderate (96 or 384-well format common) | Yes |
Surface Plasmon Resonance (SPR) | Detects changes in refractive index at a sensor surface upon antibody-antigen binding | Label-free real-time analysis, highly sensitive | Requires specialized equipment and expertise, higher cost | Low (typically single-channel, but high-throughput systems exist) | Yes |