A guide to the different techniques in which antibodies are used
A guide to the different techniques in which antibodies are used
Enzyme Linked Immunosorbent Assay or ELISA is one of the most common tests conducted in immunobiochemistry. The principle involves immobilising antibodies onto a microtitre plate. These antibodies are labelled with an enzyme; the fixed enzyme reacts with the substrate to yield a coloured product allowing the qualification and quantification of the analyte in question.
There are 2 main types of ELISA:
Competitive ELISA is principally used for the detection of small molecules, which do not have multiple epitopes available for the simultaneous binding of two antibodies.
In a competitive ELISA, antibody specific to the analyte of interest is immobilised on a micro-titre plate. Then enzyme-conjugated antigen is incubated with capture antibody in the presence of a competing, unconjugated form of the same antigen (which can be sample or calibrator). A solution containing chromophore/substrate specific to the enzyme is added. The colour intensity produced is directly proportional to the amount of conjugate enzyme bound, which in turn is inversely proportional to the amount of un-conjugated antigen present.
Sandwich ELISA is principally used for larger protein analytes, which have multiple epitopes that can be accessed by two antibodies simultaneously.
In a sandwich ELISA, antibody specific to the analyte of interest is immobilised on a micro titre plate. Then antigen is bound to the immobilized capture antibody. A second, enzyme conjugated, antibody is added, followed by a solution containing chromophore/substrate specific to the enzyme. The colour intensity produced is directly proportional to the amount of antigen present. The principal determinant in this type of ELISA is the full dynamic range of antigen over which the conjugate effectively binds.
Western Blotting can be used to determine the molecular weight of proteins and to measure the amount of a particular protein present in a sample. The proteins within a sample are first separated by electrophoresis and then transferred to a nitrocellulose or PVDF (polyvinylidene difluoride) membrane.
As a result of this process, the proteins are bound to the membrane surface allowing detection by an antibody specific to the protein of interest. However, since the membranes used have a high protein binding capacity, it is essential to block all non specific proteins. This is normally carried out by incubation of the membrane with BSA, non-fat milk powder or casein. Once non specific sites have been blocked, an antibody specific to the protein of interest can be added. This antibody can be tagged with a label that will allow subsequent
detection e.g. chemiluminescence or fluorescence. If the primary antibody does not contain a label, a secondary labelled antibody is employed.
The dot blot technique can be used to detect and identify the presence of a particular protein in a sample. The technique is very similar to the western blot technique however rather than separating the proteins in a sample using electrophoresis the sample is spotted directly onto the membrane and hybridised with an antibody probe. The method allows the concentration of a particular protein to be semi-quantitatively measured.
Immunohistochemistry (IHC) is a method of analysing and identifying protein distribution and localisation in a tissue sample based on the binding of antibodies to specific cellular targets.
For immunohistochemical analysis, it is essential that the morphology of the tissues and cells be retained and that the antigenic sites be accessible. Ideally, it would be preferable to carry out immunohistochemistry using fresh, rapidly frozen, tissue sections; however, to maintain cell integrity and preserve cell components, tissue samples are normally fixed by the addition of a chemical compound such as formalin and then embedded into wax. This fixing results in the cross linking of amino acids in the tissue and can disguise the epitope regions thereby inhibiting the action of any protein specific antibodies. Epitope recovery or retrieval is therefore required to expose hidden regions. This is usually carried out by enzymatic digestion or by the use of heat. Once tissues have been treated to remove endogenous peroxidase activity and non-specific sites have been blocked, proteins can be detected using the same methods as described for western blotting. Detection is possible through the use of either a labelled antibody specific to the protein of interest or indirectly-using an unlabelled primary antibody specific to the protein of interest followed by the addition of a secondary labelled antibody specific to the primary antibody .
Immunoprecipitation (IP) is a technique routinely used to determine the molecular weight of protein antigens, it is also used to study protein – protein interactions, to determine specific enzyme activity, to monitor post-translational modification and quantify proteins. The technique allows the detection of rare proteins which would be otherwise difficult to detect, as they can be concentrated up to 10 000 fold by immunoprecipitation.
The technique involves the purification of antibody/antigen complexes on matrices that specifically bind antibodies .
Flow cytometry is an antibody based technique used to examine the physical and chemical properties of cells. The method involves the detection of fluorescence or scattered light produced by individual cells as they flow or pass through a sensing point. Labelled antibodies allow are often used to monitor the presence of proteins and other target molecules on the surface of cells.
Flow cytometry enables the rapid analysis of several proteins on a single cell and provide quantitative data on a large number of cells as such it is frequently used in biomedical research and diagnostics to diagnose health disorders.
Fluorescence Activated Cell Sorting (FACS)
FACs is a more specialised type of flow cytometry mainly used to separate certain cell types from a mixed population of cells. Cells are separated based on their unique fluorescence or light scattering characteristics.
Enzyme Linked Immunospot (Elispot)
As the name suggests the Elispot assay is based on the ELISA methodology. The technique involves a polyvinylidene difluoride (PVDF) backed microtitre plate pre-coated in antibody specific to the analyte of interest. Under the correct conditions the capture antibody will bind to the analyte of interest.
After washing to remove any cell debris a biotinylated antibody also specific to the analyte of interest is added allowing detection of the original capture antibody. A second wash step is performed to remove any unbound antibody before adding the enzyme labelled conjugate. A coloured end product typically a black spot can then be visualised and counted. Each spot is representative of a single cell producing the analyte of interest.