Antibodies, A Laboratory Manual, Second Edition, Edited by Edward A. Greenfield



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Antibody Capture in Polyvinyl Chloride Wells: Enzyme-Linked Detection When Immunogen Is an Immunoglobulin Fusion Protein (Indirect ELISA to Detect Ig Fusion Proteins)

(Protocol summary only for purposes of this preview site)

This protocol describes the screening assay for experiments in which the immunogen is an immunoglobulin fusion protein. Because these reagents comprise the Fc region of an antibody molecule combined with the extracellular domain of a cell-surface protein, detection with a secondary alkaline phosphatase (AP)conjugated, general anti-mouse antibody will result in a positive reaction in every well. This problem can be avoided by using a secondary antibody reactive to the Fc region from a different species (e.g., human) or by switching the secondary detecting antibody to an anti-light-chain HRP antibody (kappa, lambda, or both). If the subclass of immunoglobulin used in the fusion protein is known, then a cocktail of anti-heavy-chain-specific antibodies can be made, excluding the one used in the fusion protein.

Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition

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