1. Radioimmunoassay (RIA)
It is the most sensitive technique for detecting antigen or antibody.
To determine the amount of antigen present in the test sample, a labeled antigen and a non-labeled antigen are allowed to bind competitively to a high affinity antibody. The antibody cannot distinguish between labeled from unlabeled antigen; so the two kinds of antigen compete for the available binding sites on the antibody. The labeled antigen is mixed with antibody at a concentration that saturates the antigen binding sites of the antibody. The unlabeled antigen of unknown concentration is added progressively. As the concentration of unlabeled antigen increases, more labeled antigen will be displaced from binding site. The decrease in the amount of radio-labeled antigen found to specific antibody in the presence of test sample is measured
2. Enzyme Linked Immuno-Sorbant Assay (ELISA)
ELISA has been pioneered by Engvall Perlmann and Schurs in 1972 and finally developed by Clark and Adams in1977. In ELISA, enzyme linked antibodies adsorbed on same solid surface like micro-ELISA plates having shallow wells and made up of polystyrene which has property to bind with antigen or antibody covalently are employed. Enzymes used in ELISA are alkaline phosphatase, horseradish peroxidase and β-galactosidase. ELISA assay uses a substrate which gets converted into a colored product. ELISA test is safer and less costly than RIA. It is commonly used laboratory test to detect antibodies in the blood. It involves the use of secondary antibodies. Use of biotinylated secondary antibody in ELISA increases the sensitivity of assay without compromising the specificity.
There are three types of ELISA
Indirect ELISA: Antigen is coated on the micro-titer well. Sample containing primary antibody is added and is allowed to react with the antigen attached to the well. The presence of antibody bound to the antigen is detected by adding an enzyme conjugated secondary anti-isotype antibody. The secondary antibody binds to primary antibody. After washing any free secondary antibody, a substrate for the enzyme is added that gives a coloured reaction product when measured by specialized spectrophotometric plate.
Direct ELISA: It is the method for detection of antigen. The antibody is immobilized on micro-titer well. Sample containing antigen is added and is allowed to react with immobilized antibody. The well is washed and enzyme linked antibody specific for a different epitope on antigen is added and reacts with bound antigen. After washing, the substrate is added and the coloured reaction product is measured.
Competitive ELISA: Antibody is first incubated in solution with sample containing antigen. The antigen-antibody mixture is added to antigen coated microtiter well. More the antigen present in the sample, less free antibody is available to bind to antigen coated well. Secondary antibody which is specific for isotype of primary antibody is added and it determines the amount of primary antibody. Higher the concentration of antigen in the original sample, lower is the absorbance.
3. Western Blotting
It is used for the identification of a specific protein in a complex mixture of proteins. It can also identify a specific antibody in a mixture.
Protein mixture is treated with SDS (Sodium dodecyl sulphate), which is a strong denaturing agent and it is then separated by electrophoresis in SDS -PAGE. The electrophoresis step separates the components according to their molecular weight, the protein bonds are transferred to nylon membrane and the proteins in the gel are transferred to the sheet by passage of electric current. The addition of enzyme linked antibodies detects the antigen of interest and position of antibody is visualized by means of ELISA reaction. If the protein of interest is bound by a radioactive antibody; the position on the blot can be determined by using autoradiography.
The immune-precipitation technique allows isolation of antigen of interest and provides a sensitive assay for the presence of a particular antigen in given cell or tissue type.
The cells or tissues are disrupted to form an extract that is mixed with an antibody against the antigen of interest to from a precipitate. The formation of the precipitate is dependent on the antigen concentration. If the antigen concentration is too low, it may take hours or days for precipitate formation and the immunoprecipitate formed is in small amount which is difficult to isolate. These limitations can be avoided by:
• Attachment of the antibody to a solid support such as synthetic bead. This allows collecting the antigen-antibody complex by centrifugation.
• Adding a secondary antibody specific for the primary antibody to bind antigen-antibody complexes. If secondary antibody is attached to bead and then the immune precipitation are collected by pairing a magnet against side of the tube, the immune complexes can be collected by centrifugation.
5. Flow Cytometry
The flow cytometer is used for the analysis and separation of cells stained with fluorescent antibody. It uses a laser beam and light detector to count single intact cells in suspension. Every time a cell passes the laser beam, light is deflected from the detector and this interruption of the laser signal is recorded. Those cells having a fluorescently tagged antibody bound to their cell surface antigens are excited by the laser and emit light that is recorded by a second detector system located at a right angle to the laser beam.
6. FISH (Fluorescent in situ hybridization)
FISH is a powerful technique used in the detection of chromosomal abnormalities. The high sensitivity and specificity of FISH and the speed with which the assays can be performed have made FISH a pivotal cytogenetic technique that has provided significant advances in both the research and diagnosis of haematological malignancies and solid tumours. From a medical perspective, FISH can be applied to detect genetic abnormalities such as characteristic gene fusions, aneuploidy, and loss of a chromosomal region or a whole chromosome or to monitor the progression of an aberration serving as a technique that can help in both the diagnosis of a genetic disease or suggesting prognostic outcomes. FISH can also be applied to such research applications as gene mapping or the identification of novel oncogenes or genetic aberrations that contribute towards various cancers. FISH is based on DNA probes annealing to specific target sequence of sample DNA