Department of microbiology microbial food technology group a diploma in quality assurance in microbiology diploma



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Types of immunofluorescence


There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect).

Primary (direct)


Primary, or direct, immunofluorescence uses a single antibody that is chemically linked to afluorophore. The antibody recognises the target molecule and binds to it, and the fluorophore it carries can be detected via microscope. This technique has several advantages over the secondary (or indirect) protocol below because of the direct conjugation of the antibody to the fluorophore. This reduces the number of steps in the staining procedure, is therefore faster, and can avoid some issues with antibody cross-reactivity or non-specificity, which can lead to increased background signal.

Secondary (indirect)


Secondary, or indirect, immunofluorescence uses two antibodies; the first (the primary antibody) recognises the target molecule and binds to it, and the second (the secondary antibody), which carries the fluorophore, recognises the primary antibody and binds to it. This protocol is more complex than the primary (or direct) protocol above and takes more time but allows more flexibility.

This protocol is possible because an antibody consists of two parts, a variable region (which recognizes the antigen) and an invariant region (which makes up the structure of the antibody molecule). A researcher can generate several primary antibodies that recognize various antigens (have different variable regions), but all share the same invariant region. All these antibodies may therefore be recognized by a single secondary antibody. This saves the cost of modifying the primary antibodies to directly carry a fluorophore.

Different primary antibodies with different invariant regions are typically generated by raising the antibody in different species. For example, a researcher might create primary antibodies in a goat that recognize several antigens, and then employ dye-coupled rabbit secondary antibodies that recognize the goat antibody invariant region ("rabbit anti-goat" antibodies). The researcher may then create a second set of primary antibodies in a mouse that could be recognised by a separate "donkey anti-mouse" secondary antibody. This allows re-use of the difficult-to-make dye-coupled antibodies in multiple experiments.

Limitations


As with most fluorescence techniques, a significant problem with immunofluorescence is photobleaching. Loss of activity caused by photobleaching can be controlled by reducing the intensity or time-span of light exposure, by increasing the concentration of fluorophores, or by employing more robust fluorophores that are less prone to bleaching (e.g., Alexa Fluors, Seta Fluors, or DyLight Fluors).

In general, immunofluorescence is limited to fixed (i.e., dead) samples. Analysis of structures within live cells by immunofluorescence is not possible, as antibodies cannot cross the cell membrane. As such some uses of immunofluorescence have been outmoded by the development of recombinant proteins containing fluorescent protein domains, e.g., green fluorescent protein (GFP). Use of such "tagged" proteins allows determination of their localisation in live cells.


ELISA




Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay(EIA), is a biochemical technique used mainly in immunology to detect the presence of anantibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA, an unknown amount of antigen is affixed to a surface, and then a specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal, most commonly a colour change in a chemical substrate.

Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually apolystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.



Traditional ELISA typically involves chromogenic reporters and substrates which produce some kind of observable color change to indicate the presence of antigen or analyte. Newer ELISA-like techniques utilize fluorogenicelectrochemiluminescent, and real-time PCR reporters to create quantifiable signals. These new reporters can have various advantages including higher sensitivities and multiplexing[1][2]. Technically, newer assays of this type are not strictly ELISAs as they are not "enzyme-linked" but are instead linked to some non-enzymatic reporter. However, given that the general principles in these assays are largely similar, they are often grouped in the same category as ELISAs.
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