Module 9 2021

22/03/2021

Binding assay format: bridging ELISA - principle

immobilise therapeutic

add sample/ control Ab

Rely on the multivalent nature of the ADA, to bind both capture and detection reagent (e.g., therapeutic) hence forming a bridge

add biotinylated therapeutic

add streptavidin-HRP add substrate & measure absorbance

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Binding assay format: bridging ELISA

Advantages • High throughput assay • Dual arm binding ensures high specificity • Low background (low matrix effect)

Disadvantages • Therapeutic immobilization may mask epitopes.

• Requires labelled therapeutic - labelling may mask epitopes. • May fail to detect rapidly dissociating / low affinity antibodies. • Affected by therapeutic interference (false negatives) • Affected by bridging factors e.g., rheumatoid factors or soluble ligands (false positives)

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