IMMUNOASSAYS
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 valency 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 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.
Agglutination Reactions are similar in principle to precipitation reactions in that they depend on the crosslinking of polyvalent antigens. Hemagglutination is used in blood typing. RBCs are mixed on a slide with antisera to the A or B blood group antigens. If the antigen is present on the cells, they agglutinate, forming a visible lump on the slide. Bacterial agglutination is also used to diagnose infection. Serum from a patient is serially diluted in an array of tubes to which the bacteria is added. The last tube showing visible agglutination will reflect the serum antibody titer of the patient. The titer is defined as the reciprocal of the greatest serum dilution that elicits a positive agglutination reaction. Thus if serial twofold dilutions of serum are prepared and if the dilution of 1/640 shows agglutination but the dilution of 1/1280 does not, then the agglutination tier of the patient's serum is 640.
Immunofluoresence is based on tagging antibodies with a fluorescent dye, or fluorochrome. 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 lentgh.
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.
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.
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).
Enzyme-Linked Immunosorbent Assay (ELISA): is similar in principle to RIA but uses an enzyme rather than a radioactive label. In its simplest format, called direct ELISA, a solid-phase binding support such as a polystyrene plate is used to capture the tested antigen, which is then detected with the aid of a specific enzyme-lined antibody (E-Ab). The detection involves conversion of a colorless substrate solution into an intense-colored product that can be measured directly in the reaction plate. The increase in optical density during the reaction is recorded at different time points for several sample dilutions, and the collected data is then used in conjunction with a standard curve to calculate the concentration of the antigen in the original sample.
Although E-Abs can be obtained by covalently binding enzymes to antibodies through a coupling agent such as glutaraldehyde, this method is not commonly used, because it often yields products with impaired immunological and/or enzymatic activity. A superior method to prepare fully active E-Abs is by exploting the high binding affinity of avidin for biotin. The biotinylated antibody used in this method contains on average 6-8 biotin moieties, which due to their small size do not interfer with the interaction between the antibody and the captured antigen. Once unboudn antiboyd has been rinsed form the plate, the biotin moieties are used in-situ to form stable complexes with avidin-enzymes conjugates.
A major limitation of the direct ELISA is that certain antigens like low molecular weight cytokines bind poorly to polystyrene plates. To by-pass this limitation, the plate can be pre-coated with a specific antibody that recognizes the tested antigen through epitopes that are not used by the E-Ab. Using a matched pair of antigen-specific antibodies, one for capturing and one for detection, significantly improve both the sensitivity and specificity of the assay. This format is called a Sandwich ELISA and it enables detection of picogram quantities of antigens. It is the preferred method for measuring cytokines and hormones in biological samples.
ELISPOT: is a modification of the ELISA assay. Plates are coated with antigen (capture antigen) recognized by an antibody of interest or with the antibody (capture antibody) specific for the antigen whose production is being assayed. A cell population under investigation is then added to the coated plates and incubated. The cells settle onto the surface of the plate. Any secreted molecules from the cells reactive with the capture molecules are bound by the capture molecules in the vicinity of the secreting cells, producing a ring of antigen-antibody complexes around each cell that is producing the molecule of interest. The plate is then washed and an enzyme linked antibody specific for the secreted antigen or specific for the species (e.g., goat anti-rabbit) of the secreted antibody is added and allowed to bind. Subsequent development of the assay by addition of a suitable chromogenic or chemiluminescence producing substrate reveals the position of each antibody or antigen-producing cell as a point of color or light.
The advantage of this assay is that it allows the quantitative determination of the number of cells in a population that are producing antibodies specific for a given antigen or an antigen for which one has a specific antibody. The assay has supplanted cytotoxic T cell assays to access cytotoxic T cell response since INF-α which is a marker for cytotoxic T cell responses can be be detected using this technique. The steps of ELISPOT for IFNγ are the following:
(1) precoate plate with mouse anti-human IFN-gamma
(2) cells added to plate with stimulant. IFN-gamma is released from cells.
(3) IFN-gamma binds to mouse anti-human IFN-gamma antibody on the plate
(4) cells and nonbound proteins are washed away.
(5) Bioteinylated anti-human IFN-gamma detection antibody is added and binds to IFN-gamma
(6) Streptavidin-AP is added and binds to the biotinylated detection antibody
(7) a precipitating substrate is added to wells of the plate which reacts with the AP.
Immunoprecipitation: has 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.
Western blotting is used to identify a specific protein in a complex mixture of proteins.
Sensitivity of various Immunoassays
Immunoassay Sensitivity (ug Antibody/ml)
| Precipitation | 20-200 |
| Immunofluorescence | 1.0 |
| Agglutination reactions | .006-0.3 |
| ELISA | 0.0001-0.01 |
| Radioimjmunoassay | 0.0006-.006 |
