Immuno assays

Immunodiffusion

Immunodiffusion takes advantage of the fact that antibody and antigen interact in aqueous solution to form a lattice that eventually develops into a visible precipitate. Antibodies that aggregate soluble antigens are called precipitins. Formation of an Ag-Ab lattice depends on the of both the antibody and antigen. A precipitate will not form with monovalent Fab fragments. When antigen and antibody diffuse toward one another in agar a visible line of precipitation will form. Visible precipitation occurs in the region of equivalence whereas no visible precipitate forms in regions of antibody or antigen excess. In radial immunodiffusion an antigen sample is placed in a well and allowed to diffuse into agar containing a suitable dilution of an antiserum. As the antigen diffuses into the agar, the region of equivalence is established and a ring of precipitation forms around the well. The area of the precipiten ring is proportional to the concentration of antigen. By comparing the area of the precipitin ring with a standard curve (obtained by measuring the precipitin areas of known concentration of the antigen), the concentration of the antigen sample can be determined.

Immunoelectrophoresis

Immunoelectrophoresis first separates an antigen mixture (like a sample of serum) by charge using electrophoresis. Troughs are then cut into the agar gel parallel to the direction of the electric field, and antiserum is added to the troughs. Antibody and antigen then diffuse toward toward each other and produce lines of precipitation where they meet in appropriate proportions. This technique is useful in showing whether a patient overproduces some serum protein or produces abnormally low amounts of one or more isotypes, characteristic of certain immunodeficiency diseases. The immunoelectrophoretic pattern of serum from patients with multiple myeloma, for example, would show a heavy distorted arc caused by the large amount of myeloma protein.

Immunofluoresence

In the immunofluorescence assay the test antigen or antibody may be labelled directly or indirectly by use of fluorescent dyes (fluorochromes) such as fluorscein and rhodaminine which can be coupled to the test antigne or antibodies or their immunocomplexes without destroying their specificity. Such conjugates can then be visualized in a fluorescence microscope. The most commonly used fluorescent dyes are fluorescein and rhodamine. Both dyes can be conjugated to the Fc region of an antibody molecule without affecting the specificity of the antibody. Each of these dyes absorbs light at one wave lentgh and emits light at a longer wave length. 

Immunofluosence is often used to stain cell membrane molecules or tissue sections. In direct staining, the specific antibody (primary antibody) is directly conjugated with fluorescein whereas in indirect staining the primary antibody is unlabeled and is detected with an additional fluorochrome labeled antibody such as a fluorochrome labeled anti-isotype reagent such as fluorescein labeled goat anti mouse immunoglobulin. 

Stronger signals with antibodies are achieved by using the unlabled primary antibody and then detecting it with a labeled secondary antibody which has an enzyme/marker molecule attached to it. This method sometimes referred to as indirect immunocytochemistry. Commonly used marker molecules include fluorescent dyes (for fluorescence microscopy), the enzyme horseradish peroxidase (for either conventional light microsocopy or electron microscopy) and the enzymes alkaline phosphatase or peroxidase (for biochemical detection). 

Immunofluorescence has been applied to identify a number of subpopulations of lymphocytes, notably the CD4+ and CD8+ T cell subpopulations. A major application of the fluorescent-antibody technique is the localization of antigens in tissue sections or in subcellular compartments.

Enzyme-Linked Immunosorbent Assay (ELISA): See outline

ELISPOT: See outline

Flow Cytometry and Fluorescence: 

The fluorescent antibody techniques above are valuable qualitative tools, but they do not give quantitative data. This problem was remedied by development of the flow cytometer, which automates the analysis and separation of cells stained with fluorescent antibody.

–Fluorescence-activated cell sorting (FACS)

Cell sorting can be performed using automated FACS such as a FACScanTM or BD InfluxTM, Becton Dickinson or an EPICS EliteTM or Beckman Coulter. Correlated measurements of FSC and SSC can allow for differentiation of cell types in a heterogeneous cell population. The staining pattern, e.g., fluoresence, combining with FSC and SSC data, can be used to identify which cells are present in a sample and to count their relative percentages. 

1. forward-scatter light (FSC) is propotrional to cell surface area or size. As a measurement of mostly diffracted light, FSC provides a suitable method of detecitng prticles greater than a given size independent of their fluorescence.

2. side-scattered light (SSC) is proportional to cell granularity or internal complexity, based on a measurement of mostly refracted and reflected light. 

Radioimmunoassay: 

Radioimmunoassay involves competitve binding of radiolabeled antigen and unlabeled antigen to a high affinity antibody.The labeled antigen is mixed with antibody at a concentration that saturates the antigen binding sites of of the antibody. Then test samples of unlabeled antigen of unknown concentration added in progressively larger amounts. The two kinds of antigen (labeled and unlabeled) compete for available binding sites on the antibody. As the concentration of unlabeled antigen increased, more labeled antigen will be displaced form the binding sites. This decrease is measured in order to determine the amount of antigen present in the test sample using a standard curve. A standard curve can be generated using unlabeled antigen samples of known concentration (in place of the test sample).

Immunoprecipitation:

Immunoprecipitationhas the advantage of allowing the isolation of the antigen of interest for further analysis. An extract producing by disruption of cells or tissues is mixed with an antibody against the antigen of interest in order to form an antigen-antibody complex that will precipitate. The antibody can be attached to a synthetic bead which allows the antigen-antibody complex to be collected by centrifugation. There are commercial kits that make it easy to generate immobilized antibodies. With these bead-linked antibodies, immunoprecipitations are followed by brief tratment with a denaturant (e.g., 1M acetic acid) which releases the bound protein from the beadh-linked antibody. These beads can even by magnetic so that immunoprecipitates are collected by placing a magnet against the side of the tube. 

If the target protein is associated tightly enough with another protein when it is captured by the antibody, the partner also precipitates. This method can be useful for identifying proteins that are part of a complex inside cells, including those that interact only transiently as where cells are stimulated by signal molecules.

 is used to identify a specific protein in a complex mixture of proteins.

Sensitivity of various Immunoassays

Immunoassay         Sensitivity (ug Antibody/ml)