{"id":218,"date":"2017-12-27T14:46:49","date_gmt":"2017-12-27T14:46:49","guid":{"rendered":"http:\/\/clinicalbiochemistry.net\/?page_id=218"},"modified":"2017-12-27T14:51:36","modified_gmt":"2017-12-27T14:51:36","slug":"book-chapter-elisa-development","status":"publish","type":"page","link":"http:\/\/clinicalbiochemistry.net\/?page_id=218","title":{"rendered":"Book Chapter: ELISA Development"},"content":{"rendered":"

McDowell, G<\/strong>., Body, R., Kirwan, C., Byrne, G. & Slevin, M. (2015) \u2018A Scheme for the Development and Validation of Enzyme Linked Immunosorbent Assays (ELISA) for Measurement of Angiogenic Biomarkers in Human Blood<\/strong>.\u2019 In: Slevin, M. & McDowell, G. (eds.) (2015) Handbook of Vascular Biology Techniques<\/strong><\/em><\/a>. London: Springer pp.453-463 (10 pages)<\/p>\n

 <\/p>\n

Abstract:<\/strong><\/p>\n

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.<\/p>\n

 <\/p>\n

Introduction <\/strong><\/p>\n

\"\"This handbook of methods in vascular biology reports a number of analytical methods for the quantification of angiogenesis, both in vitro<\/em> and in vivo<\/em>. 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.<\/p>\n

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.<\/p>\n

 <\/p>\n

Key words: <\/strong>ELISA, Reagents, Development, Validation, VCAM, angiogenesis markers, serum<\/p>\n","protected":false},"excerpt":{"rendered":"

McDowell, G., Body, R., Kirwan, C., Byrne, G. & Slevin, M. (2015) \u2018A Scheme for the Development and Validation of Enzyme Linked Immunosorbent Assays (ELISA) for Measurement of Angiogenic Biomarkers in Human Blood.\u2019 In: Slevin, M. & McDowell, G. (eds.)<\/p>\n","protected":false},"author":1,"featured_media":69,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"jetpack_post_was_ever_published":false,"_links_to":"","_links_to_target":""},"jetpack_shortlink":"https:\/\/wp.me\/P9tPlw-3w","jetpack-related-posts":[{"id":121,"url":"http:\/\/clinicalbiochemistry.net\/?page_id=121","url_meta":{"origin":218,"position":0},"title":"Research Papers","date":"December 22, 2017","format":false,"excerpt":"Body, R., McDowell, G., Carley, S., Ferguson, J. & Mackway-Jones, K. (2010) \u2018Diagnosing acute myocardial infarction with troponins: how low can you go?\u2019 Emerg Med J. 27 pp. 292-296. Body, R., Carley, S., Wibberley, C., McDowell, G., Ferguson, J. & Mackway- Jones, K. (2010) \u2018The value of symptoms and signs\u2026","rel":"","context":"Similar post","img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]},{"id":114,"url":"http:\/\/clinicalbiochemistry.net\/?page_id=114","url_meta":{"origin":218,"position":1},"title":"Handbook of Vascular Biology Techniques","date":"December 22, 2017","format":false,"excerpt":"The\u00a0Handbook of Vascular Biology Techniques, edited by Mark Slevin and Garry McDowell, and published by Springer, contains detailed descriptions of a variety of techniques and methods in both basic and advanced vascular biology. Methodologies range from\u00a0in-vitro\u00a0cell culture to\u00a0in-vivo\u00a0manipulations, through cell signalling proteomics and genomics to patient imaging. The book contains\u2026","rel":"","context":"Similar post","img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]},{"id":499,"url":"http:\/\/clinicalbiochemistry.net\/?page_id=499","url_meta":{"origin":218,"position":2},"title":"Acetlycholine & Monomeric C-Reactive Protein","date":"January 1, 2019","format":false,"excerpt":"Slevin, M., Lemma, R.S., Zeinolabediny, Y., Liu, D., Ferris, G.R., Caprio, V., Phillips, N., Di Napoli, M., Guo, B., Zeng, X., AlBaradie, R., Binsaleh, N.K.,\u00a0McDowell, G. & Fang, W-H. (2018) Acetylcholine inhibits monomeric C-Reactive Protein inducd inflammation, endothelial cell adhesion and platelet aggregation; A potential therapeutic. Frontiers in Immunology 9\u2026","rel":"","context":"Similar post","img":{"alt_text":"","src":"https:\/\/i0.wp.com\/clinicalbiochemistry.net\/wp-content\/uploads\/2017\/12\/Lab-Equipment-Pages.png?fit=800%2C534&resize=350%2C200","width":350,"height":200},"classes":[]},{"id":488,"url":"http:\/\/clinicalbiochemistry.net\/?page_id=488","url_meta":{"origin":218,"position":3},"title":"Book Chapter: Biophysical and molecular targets.","date":"January 1, 2019","format":false,"excerpt":"Slevin, M., Carroll, M., Murgatroyd, C. &\u00a0McDowell, G.(2014) \u2018Biophysical and molecular targets.\u2019 In: Kibos, A.S., Knight, B.P., Essebag, V., Fishberger, S.B., Slevin, M. & Tintoiu I. (eds.) (2014)\u00a0Cardiac Arrhythmias: From Basic Mechanisms to State-of-the-art Management. London: Springer pp. 335-343 (9 pages) Cardiac arrhythmias represent a large heterogeneous collection of heart\u2026","rel":"","context":"Similar post","img":{"alt_text":"","src":"https:\/\/i0.wp.com\/clinicalbiochemistry.net\/wp-content\/uploads\/2019\/01\/Cardiac-Arrhthmias-Book-Full-size-e1549719879589.png?fit=600%2C848&resize=350%2C200","width":350,"height":200},"classes":[]},{"id":136,"url":"http:\/\/clinicalbiochemistry.net\/?page_id=136","url_meta":{"origin":218,"position":4},"title":"T-MACS","date":"December 22, 2017","format":false,"excerpt":"Body, R., Carlton, E., Sperrin, M., Lewis, P.S., Burrows, G., Carley, S., McDowell, G., Buchan, I., Greaves, K. & Mackway-Jones, K. (2017). Troponin only Manchester Acute Coronary Syndromes (T-MACS) decision aid: single biomarker re-derivation and external validation in three cohorts. Emerg Med J 34 pp. 349-356 Background The original Manchester\u2026","rel":"","context":"Similar post","img":{"alt_text":"","src":"https:\/\/i0.wp.com\/clinicalbiochemistry.net\/wp-content\/uploads\/2017\/12\/Cardiac-Marker-Test-Pages-300x200.png?resize=350%2C200","width":350,"height":200},"classes":[]},{"id":619,"url":"http:\/\/clinicalbiochemistry.net\/?page_id=619","url_meta":{"origin":218,"position":5},"title":"MSCs loaded with p5","date":"January 27, 2019","format":false,"excerpt":"Mesenchymal Stem Cells Loaded with p5, Derived from CDK5 Activator p35, Inhibit Calcium-Induced CDK5 Activation in Endothelial Cells Wen-Hui Fang, Shant Kumar, Garry McDowell, David Smith, Jurek Krupinski, Peter Olah, Raid Saleem Al-Baradie, Mohammad Othman Al-Rukban, Eugene Bogdan Petcu, and Mark Slevin. The potential use of stem cells as therapeutics\u2026","rel":"","context":"Similar post","img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]}],"_links":{"self":[{"href":"http:\/\/clinicalbiochemistry.net\/index.php?rest_route=\/wp\/v2\/pages\/218"}],"collection":[{"href":"http:\/\/clinicalbiochemistry.net\/index.php?rest_route=\/wp\/v2\/pages"}],"about":[{"href":"http:\/\/clinicalbiochemistry.net\/index.php?rest_route=\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"http:\/\/clinicalbiochemistry.net\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/clinicalbiochemistry.net\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=218"}],"version-history":[{"count":2,"href":"http:\/\/clinicalbiochemistry.net\/index.php?rest_route=\/wp\/v2\/pages\/218\/revisions"}],"predecessor-version":[{"id":221,"href":"http:\/\/clinicalbiochemistry.net\/index.php?rest_route=\/wp\/v2\/pages\/218\/revisions\/221"}],"wp:featuredmedia":[{"embeddable":true,"href":"http:\/\/clinicalbiochemistry.net\/index.php?rest_route=\/wp\/v2\/media\/69"}],"wp:attachment":[{"href":"http:\/\/clinicalbiochemistry.net\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=218"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}