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LS6006 ELISA Practical

Annotated ELISA data, Graph and Answers to Questions

K number: 1458756 Date of practical: 24/10/2016

Partner K Number: 1406775

Marking Criteria are explained on assessment brief you should refer to these.

Type answers using Arial 11 point font.  Use the box line numbers below as guidance for expected answer length: do not write more. Add your KU no. and partner KU no. and other annotations to data prior to scanning. Embed annotated raw data and Excel graph into the document.

A) Your ELISA data and annotations of data will be assessed at the end of the practical. You must also scan these results and incorporate into your report (5 marks).

Note ELISA data must  be embedded into report for a pass.

B1. Draw, label, and append an excel graph of log10 concentration of antigen against mean absorbance. (4 marks)

B2. Deduce antigen concentrations in the two unknown samples U1 and U2 using for your graph to read off values. Comment on the accuracy/precision of your data and evaluate the sources of error. (3 marks)

U1 = 25 µg/ml  U2 = 0.25 µg/ml  

Due to a high value for the blank, negative results were acquired, making the graph skewed, and the results for U1 and U2 less reliable. The duplicates were not of close proximity, giving a larger range for error, and changing the mean especially for the neat solution.

B3. Briefly compare your method with a non-competitive “sandwich” ELISA:  compare the advantages and disadvantages of each method, and critically compare their analytical sensitivity and specificity. (4 marks)

In a sandwich ELISA (s-ELISA), a micro-titre plate is coated with capture antibodies (Ab), and the sample solution/ target antigens (Ag) is then added. Detection Ab are added which bind to a different epitope of the target Ag to that of the capture Ab. In the competitive ELISA (c-ELISA), which is the method used, known Ab are incubated with target Ag, and added to Ag-coated wells. The plate is washed and enzyme-conjugated Ab is added. Competition between the free and bound Ag occurs. Substrate is added as the final stage in both assays. Impure samples can be used in both assays which allows complex samples to be used, without the use of an extra step. The s-ELISA is more sensitive to matrix effects and sample dilution, which means it requires further sample processing, whereas the c-ELISA does not. The c-ELISA produces results with less irregularity between duplicates, yet is less sensitive, and specific than a sandwich assay. The analytical specificity is higher in a s-ELISA as 2 different primary Ab are used, though due to the requirement of matched pairs, not all Abs can be used for detection. John R. Crowther. (2008). The ELISA Guidebook: Second Edition (Methods in Molecular Biology). 2nd Edition. Humana Press.

B4. Briefly explain one other types of immunoassay that can be used for quantitative antigen or antibody analysis to investigate named immunological diseases. (2 marks)

Rapid immunochromatographic assay is an assay that has been used to identify and study a number of different immunological diseases including Dengue Virus Infection. It is a handheld assay that involves a labelled antibody dried onto a strip, where a capture antibody is added and the target specimen is applied; the presence of a red line on the strip is observed.

Vaughn et al., (1998) Evaluation of a Rapid Immunochromatographic Test for Diagnosis of Dengue Virus Infection. Journal of Clinical Microbiology. 36(1), 234–238.

B5. Review and evaluate one important clinical application of ELISA in the investigation of an immunological disease (not an infectious disease). Include details of the antigens & antibodies used and discuss the diagnostic sensitivity & specificity of the assay. (7 marks)

Research has been carried out to investigate the benefits of using ELISA to determine coeliac disease, a disease affecting around 1 in 100 individuals in the UK. It is characterised by an intolerance to gluten prolamins, and is considered an autoimmune disorder. In regards to diagnosis, different serological markers can be identified in a patient’s blood: including IGA antiendomysium antibodies (IgA-EmA), and IgA anti-gliadin antibodies (IgA-AGA). High levels of these antibodies indicate that the patient has the disease. Within the serum of patients with Coeliac Disease, many different anti-gliadin antibodies are found, including IgG, IgE and IgA forms, with the latter being the most abundant. The identification of such markers is carried out by different methods, with ELISA being the most widely utilized method, using the IgA-AGA marker. These antibodies are produced in response to a class of proteins known as gliadins, found in wheat. Once digested, the body produces these antibodies in response, in much higher quantities in patients with the disorder. Micro-ELISA is used to test for the presence of these antibodies, using crude gliadin as the antigen, and IgA-AGA as the primary antibody. Many different studies have shown that IgA-AGA has a higher specificity, but a lower sensitivity than that of IgG-AGA, so the IgA form is most preferred. Studies have found that the specificity of these antibodies have ~85% specificity, and ~97% sensitivity in adults. Volta, U., Lenzi, M., Lazzari, R., Cassani, F., Collina, A., Bianchi, F.B. and Pisi, E. (1985) Antibodies to gliadin detected by immunofluorescence and a micro-eLISA method: Markers of active childhood and adult coeliac disease, Gut, 26(7), pp. 667–671.

(Total Marks available 25)

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