Quantitative differences among various proteins as blocking agents for ELISA microtiter plates.

Area of Science:

• Biochemistry
• Immunology
• Assay Development

Background:

• Non-specific binding (NSB) in immunoassays, such as enzyme-linked immunosorbent assays (ELISA), can lead to false-positive results.
• Effective blocking agents are crucial for minimizing NSB and improving assay specificity.

Purpose of the Study:

• To evaluate the efficacy of various proteins as blocking agents for polystyrene microtiter plates in preventing NSB.
• To compare the performance of different blocking proteins under simultaneous incubation and pretreatment conditions.

Main Methods:

• Proteins including instantized milk, casein, pig and fish skin gelatins, and serum albumin were tested for NSB blocking ability.
• A million-fold concentration range was used for each protein.
• Blocking was assessed in simultaneous incubation with a peroxidase-conjugated immunoglobulin and as a pretreatment agent.

Main Results:

• Instantized milk and casein demonstrated over 90% NSB inhibition at low concentrations in both simultaneous and pretreatment modes.
• Enzymatically hydrolyzed porcine skin gelatin was the least effective, showing poor blocking activity, especially as a pretreatment agent.
• Fish skin gelatin exhibited better blocking than hydrolyzed porcine gelatin and remained fluid when refrigerated.

Conclusions:

• Casein and milk proteins are highly effective NSB blockers, likely through protein-plastic interactions.
• Porcine skin gelatin's blocking is primarily via protein-protein interactions, with hydrolyzed forms being less effective.
• Empirical testing is necessary for specific ELISA systems, but casein and milk offer promising candidates for blocking agents.