Monoclonal antibodies (Folder 1.3) are those produced by an antibody-producing cell line consisting of a single group (clone) of identical B lymphocytes. The single clone that becomes a cell line is obtained from an individual with multiple myeloma, a tumor derived from a single antibody-producing plasma cell. Individuals with multiple myelomas produce a large population of identical, homogeneous antibodies with an identical specificity against an antigen. To produce monoclonal antibodies against a specific antigen, a mouse or rat is immunized with that antigen. The activated B lymphocytes are then isolated from the lymphatic tissue (spleen or lymph nodes) of the animal and fused with the myeloma cell line. This fusion produces a hybridoma, an immortalized individual antibody-secreting cell line. To obtain monoclonal antibodies against rat actin molecules, for example, the B lymphocytes from the lymphatic organs of immunized rabbits must be fused with myeloma cells.
FOLDER 1.3 Clinical Correlation: Monoclonal Antibodies in Medicine
Monoclonal antibodies are now widely used in im-munocytochemical techniques and also have many clini-cal applications. Monoclonal antibodies conjugated with radioactive compounds are used to detect and diagnose tumor metastasis in pathology, differentiate subtypes of tumors and stages of their differentiation, and in infec-tious disease diagnosis to identify microorganisms in blood and tissue fluids. In recent clinical studies, mono-clonal antibodies conjugated with immunotoxins, chemotherapy agents, or radioisotopes have been used to deliver therapeutic agents to specific tumor cells in the body.
Both direct and indirect immunocytochemical methods are used to locate a target antigen in cells and tissues.
The oldest immunocytochemistry technique used for identifying the distribution of an antigen within cells and tissues is known as direct immunoﬂuorescence. This technique uses a fluorochrome-labeled primary antibody (either polyclonal or monoclonal) that reacts with the antigen within the sample (Fig. 1.5a). As a one-step procedure, this method involves only a single labeled antibody. Visualization of structures is not ideal because of the low intensity of the signal emission. Direct immunofluorescence methods are now being replaced by the indirect method because of suboptimal sensitivity.
Indirect immunoﬂuorescence provides much greater sensitivity than direct methods and is often referred to as the “sandwich” or “double-layer technique.” Instead of conjugating a fluorochrome with a specific (primary) antibody directed against the antigen of interest (e.g., a rat actin molecule), the fluorochrome is conjugated with a secondary antibody directed against rat primary antibody (i.e., goat anti-rat antibody; Fig. 1.5b). Therefore, when the fluorescein is conjugated directly with the specific primary antibody, the method is direct; when fluorescein is conjugated with a secondary antibody, the method is indirect. The indirect method considerably enhances the fluorescence signal emission from the tissue. An additional advantage of the indirect labeling method is that a single secondary antibody can be used to localize the tissue-specific binding of several different primary antibodies (Fig. 1.6). For microscopic studies, the secondary antibody can be conjugated with different fluorescent dyes so that multiple labels can be shown in the same tissue section (see Fig. 1.4). Drawbacks of indirect immunofluorescence are that it is expensive, labor intensive, and not easily adapted to automated procedures.
It is also possible to conjugate polyclonal or monoclonal antibodies with other substances, such as enzymes