McDowell, G., Body, R., Kirwan, C., Byrne, G. & Slevin, M. (2015) ‘A Scheme for the Development and Validation of Enzyme Linked Immunosorbent Assays (ELISA) for Measurement of Angiogenic Biomarkers in Human Blood.’ In: Slevin, M. & McDowell, G. (eds.) (2015) Handbook of Vascular Biology Techniques. London: Springer pp.453-463 (10 pages)
Abstract:
In this chapter the authors describe a protocol that can be applied to design and validate an ELISA technique using commercially available reagents. This often proves an economical alternative to purchasing off-the-shelf-kits. In addition, this protocol allows custom validation of the ELISA to a specific purpose. We have illustrated our protocol using VCAM as an example, however the principles and techniques described can equally be applied to all ELISA based techniques.
Introduction
This handbook of methods in vascular biology reports a number of analytical methods for the quantification of angiogenesis, both in vitro and in vivo. In this chapter the authors discuss a scheme for the development and validation of ELISA for the quantification of circulating vascular biomarkers. The principles we discuss, can be applied to the majority of ELISA techniques, irrespective of the biomarker being measured. We will illustrate our method using Vascular Cell Adhesion Molecule-1 (VCAM-1) as an example.
The ELISA is an example of a non-competitive sandwich assay. The components of the ELISA consist of a capture antibody, secondary detection antibody and detection reagent. Briefly, an analyte specific capture antibody is bound to an ELISA plate, forming the solid phase. The analyte of interest in samples, or standards is then incubated with the solid phase antibody, capturing the analyte in the solid phase, due to the antibody-antigen reaction. The plate is washed to remove unbound analyte. Following the wash step, a secondary biotin-conjugated antibody is added. The secondary antibody binding to different epitopes on the antigen to the capture antibody. Following a wash step to remove unbound secondary antibody, a Streptavidin-horseradish peroxidase enzyme conjugate is added. Streptavidin binds biotin with high affinity. Again following a wash step to remove unbound enzyme conjugate a substrate solution is added, which changes colour in the presence of the enzyme. The reaction can be stopped and the colour intensity (optical density) measured. In the non-competitive format, optical density is directly proportional to concentration.
Key words: ELISA, Reagents, Development, Validation, VCAM, angiogenesis markers, serum
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