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

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

Goal setting as an outcome measure: a systematic review

Background: Goal achievement has been considered to be an important measure of outcome by clinicians working with patients in physical and neurological rehabilitation settings. This systematic review was undertaken to examine the reliability, validity and sensitivity of goal setting and goal attainment scaling approaches when used with working age and older people. Aims and objectives: To review the reliability, validity and sensitivity of both goal setting and goal attainment scaling when employed as an outcome measure within a physical and neurological working age and older person rehabilitation environment, by examining the research literature covering the 36 years since goal-setting theory was proposed. Methods: Data sources included a computer-aided literature search of published studies examining the reliability, validity and sensitivity of goal setting/goal attainment scaling, with further references sourced from articles obtained through this process. Main findings: There is strong evidence for the reliability, validity and sensitivity of goal attainment scaling. Empirical support was found for the validity of goal setting but research demonstrating its reliability and sensitivity is limited. Conclusions: Goal attainment scaling appears to be a sound measure for use in physical rehabilitation settings with working age and older people. Further work needs to be carried out with goal setting to establish its reliability and sensitivity as a measurement tool