Module 9 2024

03/09/2024

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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