containing such gene fusions are used to infect bacteria, and the resulting phage particles have coats that express the antibody-like fusion protein, with the antigen-binding domain displayed on the outside of the bacteriophages. A collection of recombinant phages, each displaying a different antigen-binding domain on the surface, is known as a phage display library. In much the same way that antibodies specific for a particular antigen can be isolated from a complex mixture by affinity chromatography (see Section A-3), phages expressing antigen-binding domains specific for a particular antigen can be isolated by selecting the phages in the library for binding to that antigen. The phage particles that bind are recovered and used to infect fresh bacteria. Each phage isolated in this way will produce a monoclonal antigen-binding particle analogous to a monoclonal antibody (Fig. A.10). The genes encoding the antigen-binding site, which are unique to each phage, can then be recovered from the phage DNA and used to construct genes for a complete antibody molecule by joining them to parts of immunoglobulin genes that encode the invariant parts of an antibody. When these reconstructed antibody genes are introduced into a suitable host-cell line, such as the non-antibody-producing myeloma cells used for hybridomas, the transfected cells can secrete antibodies with all the desirable characteristics of monoclonal antibodies produced from hybridomas.
A-9 Generation of human monoclonal antibodies from vaccinated individuals.
In some cases, human monoclonal antibodies can be made by cloning the rearranged antibody heavyand light-chain gene sequences from plasma cells isolated from vaccinated individuals. Based on the expression of cell-surface molecules such as CD27 and CD38, human plasma cells can be isolated from the peripheral blood of individuals who were immunized approximately 1 week earlier. Individual plasma cells are sorted into wells of microtiter plates, and the antibody heavyand light-chain variable-region sequences are cloned from each cell by PCR. These sequences are then inserted into constructs that recreate the full-length antibody heavyand light-chain genes, and the paired heavyand light-chain vectors are introduced into an immortalized human cell line. Human cells are then screened to identify those secreting antibody proteins that bind to the immunizing antigen. These immortalized cells become a permanent source of the specific human antibody protein.
A-10 Microscopy and imaging using fluorescent dyes.
Because antibodies bind stably and specifically to their corresponding antigen, they are invaluable as probes for identifying a particular molecule in cells, tissues, or biological fluids. Antibody molecules can be used to locate their target molecules accurately in single cells or in tissue sections by a variety of different labeling techniques. When the antibody itself, or the anti-immunoglobulin antibody used to detect it, is labeled with a fluorescent dye (a fluorochrome or fluorophore) and then detected by microscopy, the technique is known as immunofluorescence microscopy. As in all serological techniques, the antibody binds stably to its antigen, allowing unbound antibody to be removed by thorough washing. Because antibodies against proteins recognize the surface features of the native, folded protein, the native structure of the protein being sought usually needs to be preserved, either by using only the most gentle chemical fixation techniques or by using frozen tissue sections that are fixed only after the antibody reaction has been performed. Some antibodies, however, bind proteins even if they are denatured, and such antibodies will bind specifically even to protein in fixed tissue sections.