Antibody Labeling Procedures

ELISA

ELISA, or enzyme-linked immunosorbent assay, is a biological laboratory technique used to detect the presence of an antigen or antibody. It is often used in medical diagnosis to determine whether a patient has been exposed to a particular type of infection.



To perform an ELISA, the sample—containing the antigen of interest—is anchored to a solid support in a dish. A solution with the complementary antibody is added to the dish, and the antibody binds to the antigen. Excess antibody is then washed away, leaving only the antigen-antibody-bound pairs in the dish. These can be visualized by adding a fluorescent molecule that binds to the antibody and gives off a visual signal, which can then be quantified. In this way, the presence and amount of the antigen of interest can be indirectly determined.

Western Blot

A Western blot is a different type of technique used to detect a specific protein in a sample and determine its size. First, sample proteins are spread out by size on a gel using gel electrophoresis. At this point, proteins are not visible to the naked eye. Scientists then transfer the proteins to a membrane and add a solution containing the antibody to the antigen of interest. After washing away excess solution, the antibody is bound to the antigen on the membrane.



Adding a secondary antibody causes the original, or primary, antibody to emit light. Quantifying the amount of light emitted allows researchers to determine the presence of the antigen, its size compared to other proteins and its relative concentration.

Immunohistochemistry

Immunohistochemistry is a technique that allows researchers to visualize the location of a protein in a tissue sample. Small slices of a sample are prepared and a primary antibody, which attaches to the antigen of interest, is added. Excess solution is washed away before researchers add a secondary antibody. This antibody attaches to the primary antibody-antigen pair and emits light, signalling the location of the antigen of interest.



Scientists use a microscope to visualize where the antigen is in the cell. Multiple different antibodies, each specific to a particular protein, can be used to determine the relative location of several molecules in a cell. If this is the goal, different colored secondary antibodies are used to distinguish between the different molecules of interest.

Flow Cytometry

Fluorescence-activated cell sorting—or FACS—is a type of flow cytometry used to separate different types of cells in a solution. Each different type of cell is "tagged" with an antibody specific to that cell type. Each of these antibodies emits a different color of light. A machine agitates the solution to break it into individual droplets and uses a sensor to detect the color of each droplet. The machine sorts the cells by emitted color, dividing them based on the type of protein they contain. FACS methodology allows researchers to sort and quantify cells of interest to their research questions.

Immuno-Electron Microscopy

Normal electron microscopy allows scientists to examine the structure of a cell magnified up to 1 million times. Immuno-electron microscopy uses the properties of antibody-antigen binding to visualize particular proteins in very thin tissue sections. First, antibodies with attached gold particles are allowed to bind to the antigen of interest. An electron microscope then visualizes the gold particles, giving researchers an image of where the protein is localized in the cell.



Although immuno-electron microscopy is simple in theory, it is technically difficult and very expensive to employ, limiting its popularity of use in many biological research programs. Antibody labeling procedures are techniques used in the biological sciences to detect the presence of specific molecules. These procedures make use of the relationship between an antigen and antibody. Antigens can be any molecule that the immune system recognizes. An antibody is a Y-shaped protein that specifically recognizes a single antigen. Biologists use the binding relationship between an antibody and antigen to determine the location of a specific molecule in a sample.