Week 9 Antibody based techniques PDF

Preview

Summary

Module run by Dirk....

Description

Antibody based techniques Antibodies are proteins made up from 4 polypeptide chains (quaternary structure). Consists of two heavy chains and two light chains. They are

binding proteins that recognise structure that are foreign and need to be removed. Immunoglobulin G: Tetrameric protein – two heavy chains (~50 kDA) and two light chains (~25kDA) – joined by disulphide bonds. When denatured disulphide bonds break. Other antibodies such Immunoglobulin M, immunoglobulin A, Immunoglobulin E etc. Antibodies for analytical science  Identification of molecular target (antigen) – proposed analytical method, preparation/purification  Production of antibody – Polyclonal or Monoclonal  Purification and validation of antibody – Protein extraction, specificity and sensitivity  Different analytical techniques They can be used in laboratory research analytical techniques. They are highly specific and bind to specific samples and make them useful. If a marker is present on a sample the antibody can be used in this case. The antigen is injected into an animal with activants (triggers the immune system to react to the antigen) and the antibodies can then respond. The protein is extracted and then the antibody is tested on is specificity and sensitivity. Does it recognize the antigen you want to target or other molecules that are similar and produce false results? Antibodies can be made against any part of the antigen however need to be prepared carefully as it may target a specific region of the protein rather than the entire protein. Monoclonal and Polyclonal antibodies comparison Polyclonal Antibodies Monoclonal Antibodies Inexpensive to produce Expensive to produce Skills required for production are Training is required for the low technology used Relatively quick to produce Hybridomas take a relatively long time to produce Generate large amounts of nonGenerate large amounts of specific specific antibodies antibodies Recognize multiple epitopes on Recognize only one epitope on an any one antigen antigen Can have batch-to-batch variability Once a hybridoma is made, it is a constant and renewable source No or low batch-to-batch variability Immunoblotting (Western blot)  Gel electrophoresis - widely used method for separation of proteins (by size).

Problem: protein staining is unspecific resulting in large number of bands (for complex samples), each often representing more than one protein.  In order to react proteins with antibodies these need to be transferred from the gel onto the surface of a membrane (nitrocellulose or polyvinyl). Staining using blotting is useful as the antibody binds to the protein of interest, now the batch intensity can be quantified using a fluorescent dye. 

Steps: 1) Transfer (passive or electroblotting) 2) Blocking (to prevent unspecific binding) 3) Primary antibody (binding to target protein) 4) Enzyme-labelled secondary antibody (binding to primary antibody) 5) Substrate reaction 6) Development The primary antibody binds to the antigen at the constant region, making it easy for the antibody to be specific to all antigens of the same species of the animal being tested. The secondary antibody then binds the variable region which differs between different animals of the same species. An enzyme attached to the secondary antibody carries out a substrate reaction and a product is made that is coloured that can be detected by its quantity. ELISA

Enzyme-linked immunosorbent assay. A quantitative method, widely used in analytical sciences and medical diagnostics. Different designs exist.

Direct assay: primary antibody binds to the antigen and the enzyme catalyses the substrate reaction to make a coloured product Indirect assay: a secondary antibody binds to the primary antibody and is associated with an enzyme that also catalyses a reaction Capture assay: a capture antibody on the surface allows for the antigen to specifically bind to it. The advantage of sandwich is that the capture antibody captures the antigen on its surface rather than any random part of the surface. Steps: 1) Coat plate with a purified antigen or capture antibody 2) Incubate with sample containing antibody or antigen analyte 3) Incubate with enzymelabelled secondary antibody 4) Substrate reaction 5) Measure absorbance Immuno-biosensor

The assay is measured qualitatively however can be quantitative too. Similarly, to a pregnancy test a control line is produced and then test line produced signifies a positive result. Immunohistochemistry Staining of molecules on/in cells or tissues using labelled antibodies. It can be done on live cells (in vivo). Antibodies can be used to stick a colour to target tissue and make it more visible....