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Immunology Practical: Enzyme Linked Immunosorbent Assay (ELISA)

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Introduction

Immunology Practical: Enzyme Linked Immunosorbent Assay (ELISA) Aim To acquire understanding and knowledge to determine the antibody titre of the Rabbit Anti-Ferritin Antibody using the Enzyme Linked Immunosorbent Assay (ELISA) Introduction ELISA is a rapid immunochemical assay that involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules). The ELISA is a fundamental tool of clinical immunology, and is used as an initial screen for pathogens detection. Based on the principle of antibody-antibody interaction, in this assay you can easy visualisation the results and can be completed without the use of radioactive materials. The ELISA technique is the first and most basic assay to determine if an individual is positive for a selected pathogen, such as HIV. The applications of immunoassays are extended to other fields such as infectious diseases, autoimmune diseases, cancer, degenerative diseases, haematology and pharmacology. Applications have not been confined to human health care. Many applications have been described in veterinary medicine, agriculture, environmental health and the food industry. The assay is performed in a microtitre plate (plastic) which contains an 8 x 12 matrix of 96 wells. They also called indirect ELISA or sandwich ELISA. Microtitre plates are coated with antigen. ...read more.

Middle

Control 100�l of diluting buffer 100�l of 2� Ab H Once you have added all the solutions into the wells, you then place the plate into the incubator at 37�C for 30 minutes again covering the plate. After the 30 minutes are over, you now wash the plate again with diluting buffer 3 times, but this time you are also using distilled water using the same methodology as before. Now you need to add 100�l of urea; the substrate to all the appropriate wells as mentioned in the table below: Table3 Well's Columns Well's Row 1 2 3 4 5 6 7 8 9 10 11 12 A Add 100�l of UREA (Substrate) 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " B 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " C D 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " E 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " 100�l " F G Control 100�l " 100�l " H You then incubate the plate for 24 hour at 37�C. ...read more.

Conclusion

In wells D3, 6, 7, 8 there is slight contamination. D3 D6 D7 D8 The reason why we know that this is a contamination is because there is no positive reaction in the first wells (D1 or E1; the highly concentrated wells). Limitations to ELISA: The drawback to the antigen-coated plate method is that it is precise; limiting quantity of antigen must be bound to the microtitre plate. Furthermore any dissociated antigen will also act as an inhibitor. Reference: 1) Definition of Enzyme-linked immunosorbent assay (ELISA) http://www.medterms.com/script/main/art.asp?articlekey=9100 [accessed on the 20 Nov 2008 at 13:52] 2) G.R. Bullock, D. Van Velzen, M.J. Warhol. (1991), Volume 2. Techniques in diagnostic pathology / edited by G.R. Bullock, D. van Velzen and M.J. Warhol. Vol.2, ELISA techniques: new developments and practical applications in a braod field / volume editor, P. Herbrink. London: Academic Press. 3) Introduction to ELISA Activity. The Biology Project, The University of Arizona, Wednesday, May 3, 2000. http://www.biology.arizona.edu/IMMUNOLOGY/activities/elisa/main.html. [Accessed on 27th November 2008]. 4) Kemeny, D. M. (1991). A practical guide to ELISA / D.M. Kemeny. Oxford: Pergamon 5) Lequin RM. Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA). Clin Chem 2005;51:2415-2418 6) The Wellcome Foundation Ltd, United Medical and Dental School of Guy's and St Thomas's Hospitals. (1988). ELISA and other solid phase immunoassays: theoretical and practical aspects / edited by D.M. Kemeny and S.J. Challacombe. ChichesterbWiley. ?? ?? ?? ?? - 1 - ...read more.

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