“to demonstrate practical skills involved in the investigation of immunological disease”.

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“to demonstrate practical skills involved in the investigation of immunological disease”.

For the Results Data above use Excel to draw and label a graph of log10 soluble antigen concentration against absorbance. Add graph to this document. Show where U1 and U2 have been read from the line of best fit on the graph.

 

Clinical immunology and medical microbiology

This assignment enables students to attain LS6006 Learning Outcomes

Demonstrate safe practical skills involved in the investigation of immunological and microbiological disease and critically evaluate the theoretical and practical basis of procedures employed in the clinical immunology and diagnostic medical microbiology laboratories.

Evaluate relevant laboratory data; write coherent and analytical laboratory reports and demonstrate skills in communication and independent learning “to demonstrate practical skills involved in the investigation of immunological disease”.

Introduction to ELISA

Antibodies can be used in many highly specific and sensitive assays to detect and quantify antigens. These require a specific antibody for the antigen to be assayed and a means of detecting antigen/antibody interaction. The Enzyme-Linked Immunosorbent Assay (ELISA) uses an enzyme directly conjugated to an antibody which produces a coloured product from a colourless substrate. Examples of such enzymes include horseradish peroxidase and alkaline phosphatase.

The basic principle of the assay involves 3 steps. Firstly antigen is bound to a surface normally the well of a micro-titre plate. Secondly enzyme-linked antibody is added to the well allowed to bind to the antigen and then unbound antibody is washed off. The substrate is then added and the amount of colour produced is proportional to the amount of antigen present.

Many variations of this method allow detection of antibody or antigen and can be used to quantify the concentration of an unknown sample. In this example a competitive ELISA is used to quantify the concentration of antigen in two samples.

In a competitive ELISA antigen is bound to the micro-titre plate as before but at the second stage a known concentration of soluble antigen is added at the same time as the enzyme-linked antibody. There is competition for antibody binding between the soluble antigen and the bound antigen. The more soluble antigen present the less antibody can bind and the less colour is produced in the third step. Therefore the amount of colour that develops is inversely proportional to the amount of soluble antigen present.

In this example you are given two samples of unknown antigen concentration which you need to quantify by reference to a standard curve.

YOUR ELISA RESULTS DATA: complete table and plot graph

Concentration Abs1 Abs2 Abs1 – Background Abs2 – Background  Mean
Units : µg/mL A450 A450 A450 A450 A450
Background (A) 0.048 0.052
10 0.080 0.076
1 0.094 0.094
0.1 0.101 0.099
0.01 0.129 0.132
0.001 0.147 0.149

U1 0.112 0.110
U2 0.098 0.101
ANSWER THE FOLLOWING QUESTIONS IN THE BOXES PROVIDED AND SUBMIT THESE PAGES PLUS THE EMBEDDED GRAPH AND THE COMPLETED FRONTSHEET (Page 1)

Type your answers in Arial 11 font. Your answers are expected to fill the table line count. Submit questions and answers (not Introduction or methods). Marking criteria are in red and marks available in bold (Total 25 Marks).

  1. For the Results Data above use Excel to draw and label a graph of log10 soluble antigen concentration against absorbance. Add graph to this document. Show where U1 and U2 have been read from the line of best fit on the graph. 5 Marks
  2. Read off the U1 and U2 antigen concentrations and place in table below with units). Comment on the precision of the data and evaluate any sources of error. 4 Marks

U1 =
U2 =

  1. Briefly compare this competitive ELISA method with a non-competitive “sandwich” ELISA. Include the advantages and disadvantages of each method and critically compare their sensitivity and specificity 5 Marks

Key Reference (Harvard):

  1. Briefly explain three other types of immunoassays (not ELISA) that can be used for quantifying antigen or antibodies to investigate immunological diseases. 3 Marks

a

b

c

  1. Critically evaluate one important clinical application of ELISA in the investigation of a named infectious disease. Include details of the antigens & antibodies used and the analytical and diagnostic sensitivity & specificity of the assay. 8 Marks

Key Reference (Harvard):