Enzyme-Linked Immunosorbent Assay (ELISA)
Enzyme-linked immunosorbent assays (ELISA) depend upon the occurrence of antibody/antigen binding to quantify and characterize specific interactions between molecules as well as substances to be analyzed.
In this procedure, antibodies that target the molecule or material to be studied are linked to a reporter enzyme in a process called conjugation. Once the enzyme substrate is added, the enzyme acts upon it to generate a molecule which produces a change in color. The reaction can be quantified with the help of a spectrophotometer to indicate how much of the antigen is concentrated within the sample.
ELISA testing has many formats, and the choice of which one to use will vary with factors such as the level of sensitivity and specificity required, as well as the assay time. The SimpleStep ELISA™ testing kits from Abcam are superior to standard ELISA kits in that they provide the same sensitivity and level of reliability but run twice as fast, so that the results are available in only half the time – but at the same cost as an ordinary kit.
Western blot (WB) testing is often chosen when the protein levels of multiple samples need to be compared relative to each other, or the molecular weight of the molecule targeted by the antibody is to be determined while studying the post-translational processing.
In this procedure, a mixture of proteins resulting from the lysis of cells or tissues are subjected to gel electrophoresis in order to separate them by molecular weight. The next step is to transfer the separated proteins to a membrane and then allow them to react with applied antibodies, in order to study the particular protein that is under investigation.
Immunohistochemistry (IHC) is another technique in which antigen locations are sought to be defined, whether specific to a particular tissue or within a cell. These studies do not allow precise quantification of the antigen, but they do allow the level of protein expression in intact tissue to be determined better than the other two techniques.
In this procedure, antibodies which bind to a protein target are used along with antigen-antibody complexes, which are inserted into the reaction either by conjugating them with the primary antibody or with a secondary antibody. These complexes can be imaged as they bind to substrates which respond by producing a color change (chromogenic), radioactivity, or fluorescence. This is termed IHC staining.
Many methods are used to prepare and visualize the sample for IHC, and any of them may be used depending on the specimen being analyzed and the level of sensitivity desired.
Immunocytochemistry (ICC) is another technique which can help understand the pattern of distribution of proteins within a cell, using antibodies labeled with fluorescent tags. This provides higher spatial resolution than IHC because it is applied to cultured cells, which are not surrounded by a complex milieu of other cells, as cells in tissue samples would be.
Here, antibodies that react with the protein of interest are applied to a sample from a cell culture after it has been fixed and permeabilized. Fluorophores are conjugated directly to the primary antibodies or to secondary conjugated antibodies, and they help to visualize the antibody-protein binding. This produces a signal which can be seen using a microscope. When multiple primary antibodies are used with different labels, several protein targets can be analyzed simultaneously.
Flow Cytometry and FACS
Flow cytometry helps quantify some selected parameters relating to the chemical and physical properties of a cell of interest. These may include the size of the cell, or the manner of expression of markers on the cell surface and inside the cell.
In principle, flow cytometry is based on the use of labeled antibodies that target certain cells within a nonspecific group of cells. These produce fluorescence on binding the target cells. Fluorescence-activated cell sorting (FACS) is an advanced modification of flow cytometry in which the amount of fluorescence is measured and used to pick out the targeted cells from the mixed group that possesses the preset parameters such as a selected level of intensity of fluorescence, cell size, and markers of viability.
Immunoprecipitation (IP) is a very useful method of antibody analysis which can be applied to a range of discrete proteins or those in complex form, to isolate and purify them.
For this purpose, the antibodies are first bound to solid-phase substrates such as agarose or magnetic beads, which immobilize them. The beads take up antigens from more complex solutions.
A variation on this basic procedure is chromatin IP (ChIP), which helps detect the binding of a specific protein to a selected sequence of DNA in vivo.
Enzyme-linked immunospot (ELISPOT) is primarily useful to detect the presence of secreted proteins such as growth factors or cytokines, and thus help quantify and compare the immune responses that occur with a range of different stimuli.
The cells are cultured in 96-well format plates that have nitrocellulose membranes or antibody-coated PVDF. The antibody is bound by the secreted protein. The presence of any antigen-antibody interaction is determined by the addition of a secondary antibody, and the protein is seen as a spot color underlying the cell which secreted it. Thus a single spot detects a single secreting cell. The membranes are scanned and the images analyzed to provide a quantitative assessment of the percentage or the number of such secreting cells responsible for the presence of the protein.
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