Question

Immunoassays can use polyclonal or monoclonal antibody preparations.

a) How do polyclonal antibodies differ from monoclonal antibodies?
(Marks: 2)
b) Describe one immunoassay that requires the use of polyclonal antibodies.
(Marks: 4)
c) Describe one immunoassay that requires the use of monoclonal antibodies.
(Marks: 4)

Answer 1

a) The antibodies that are secreted by different B cell lineages within the body are called polyclonal antibodies. These are the group of imunoglobulin molecules molecules which react against a specific antigen each molecule identifying a different epitope.

The antibodies that are made by identical immune cells which are clones of a unique parent cell are called monoclonal antibodies. These have a monoclonal affinity in which they bind to the same epitope.

The other difference is, polyclonal antibodies are produced in live animals while monoclonal antibodies are produced ex-vivo using tissue culture techniques.

b) Polyclonal antibodies are used as a secondary antibodies in immunoassays like ELISA, western blotting, immunohistochemistry, microarray assays,flowcytometry.

ELISA is one example of immunoassay that requires the use of polyclonal antibodies. ELISA stands for Enzyme Linked Immunosorbent assay which is comonly used to measure antibodies, antigens, proteins and glycoproteins in various biological samples.

ELISAs combine the specificity of antibodies with the sensitivity of simple enzyme assays. This is done by using antibodies or antigens coupled to an easily assayed enzyme. Two varaiations are available in ELISA, one is to detect the presence of antigens that are recognized by antibody and the other is to test antibodies that recognize an antigen. It provides an useful measurement of Antigen-Antibody concentration.

There are four different types in ELISA , 1. Direct ELISA, 2. Sandwich ELISA, 3. Competitive ELISA, 4. Reverse ELISA. There are different enymatic markers used in ELISA. the comonly used are, OPD, TMB, ABTS, PNPP. ELISA is a useful tool for determining serum antibody concentrations. It is also useful in food industry to detect potential food allergens. It is also useful in toxicology as a rapid presumptive screen for certain drug classes.

c) Monoclonal antibodies can serve to detect or purify a substrate and has become an important tool in biochemistry, molecular biology and medicine. Monoclonal antibodies are used in immunoassays like western blot, immunodot blot test etc.

Western blot is one example of immunoassay that requres the use of monoclonal antibodies. However polyclonal antibodies are also used in western blotting technique.

Western blot is used for the quantitative detection of single proteins and protein modifications. It is a general method used to identify the presence of a specific single protein within a complex mixture of proteins. The size and color intensity of a protein band on the blot membrane derives the semi quantitative estimation of a protein.

Its method is composed of gel electrophoresis followed by an electrophoretic transfer onto a membrane and imunostaining procedure.

This technique is used usually for verification of protein production after cloning. It is used in medical diagnostics like HIV test or BSE test etc. Its applications also include use by world anti doping agency (WADA).