Impact of Immunization Technology and Assay Application on Antibody Performance – A Systematic Comparative Evaluation

Abstract

Antibodies are quintessential affinity reagents for the investigation and determination of a protein's expression patterns, localization, quantitation, modifications, purification, and functional understanding. Antibodies are typically used in techniques such as Western blot, immunohistochemistry (IHC), and enzyme-linked immunosorbent assays (ELISA), among others. The methods employed to generate antibodies can have a profound impact on their success in any of these applications. We raised antibodies against 10 serum proteins using 3 immunization methods: peptide antigens (3 per protein), DNA prime/protein fragment-boost (“DNA immunization”; 3 per protein), and full length protein. Antibodies thus generated were systematically evaluated using several different assay technologies (ELISA, IHC, and Western blot). Antibodies raised against peptides worked predominantly in applications where the target protein was denatured (57% success in Western blot, 66% success in immunohistochemistry), although 37% of the antibodies thus generated did not work in any of these applications. In contrast, antibodies produced by DNA immunization performed well against both denatured and native targets with a high level of success: 93% success in Western blots, 100% success in immunohistochemistry, and 79% success in ELISA. Importantly, success in one assay method was not predictive of success in another. Immunization with full length protein consistently yielded the best results; however, this method is not typically available for new targets, due to the difficulty of generating full length protein. We conclude that DNA immunization strategies which are not encumbered by the limitations of efficacy (peptides) or requirements for full length proteins can be quite successful, particularly when multiple constructs for each protein are used.

An Introduction to B-Cell Epitope Mapping and In Silico Epitope Prediction

“…Use of epitope mapping in the drug development is reviewed earlier [27]. In spite of advances in B-cell epitope mapping, it is important to note that antibodies raised against peptides often lack the ability to bind native proteins due to unstructured nature of the peptide [28]. …”

Antibody and Antigen Contact Residues Define Epitope and Paratope Size and Structure

“…For decades, researchers have attempted to use peptides as surrogates of full-length native proteins with little success (2). Anti-peptide Abs react well to their cognate peptides and may be useful for detecting linear epitopes in techniques like Western blot, but Abs raised to unstructured peptides more often than not do not recognize the same sequence in native protein (3,4).…”

Two potential therapeutic antibodies bind to a peptide segment of membrane-bound IgE in different conformations

“…NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4139 ARTICLE with the native proteins when the peptide is flexible within the native structure-for example, antibodies against peptide segments of hemagglutinin 32 and lysozyme 33 -they generally bind to the denatured protein 34 . In contrast, h4B12 and h47H4 bind to the same CemX peptide segment of native mIgE in quite different conformations with high affinity and specificity.…”