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Related Experiment Videos

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

R F Vogt, D L Phillips, L O Henderson

    Journal of Immunological Methods
    |July 16, 1987
    PubMed
    Summary

    Instantized milk and casein effectively block non-specific binding (NSB) in immunoassays, outperforming other proteins. These findings aid in selecting optimal blocking agents for ELISA systems.

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    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.

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    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.