Monoclonal antibody screening

Monoclonal antibody screens on grasshopper embryos uncovered a protein which, like Fasciclin I, II and III, is expressed on subsets of axons in the embryonic CNS. It was originally christened Fasciclin IV, but has more recently been renamed Semaphorin, as it has become clear that it differs in a number of fundamental respects from the Fas-ciclins (Kolodkin et al., 1992). The semaphorins are discussed in Section 5.2.1. 

Figure 21.1. A dot blot used in a monoclonal antibody screen (Fehon et al. 1994). Five positive hybrido-ma supernatants are apparent (encircled), while the rest had no response to the fusion protein. The nitrocellulose squares for this dot blot were not arrayed to allow multichannel pipetting of primary supernatants.

The dot blot will provide a qualitative estimate of antibody titer (Protocol 21.3). The same test can be done quantitatively by ELISA (enzyme-linked immunosorbent assay), but the technically simpler dot blot gives adequate information, uses less protein, and does not require an ELISA plate reader. If the same protein has been expressed in two different fusion-protein vectors, the test should be performed with the protein not used for immunization. If there is only one fusion protein, e.g., a GST-fusion, then a second dot blot can be done in parallel with just GST alone to compare the titers. If the antiserum recognizes the protein, there should be a lower titer of antibodies against GST alone as compared to against the entire fusion protein. A preimmune control is always advisable. Figure 21.1 shows a sample dot blot from a monoclonal antibody screen. 

I direct fluorescent monoclonal antibody test. Given the fact that most women are asymptomatic, an annual screening or physical is necessary, as early detection may reduce rates of transmission. 

Many variations on the assay exist, but the ELISA, shown schematically below, is currently highly favored because of its simplicity once established in a laboratory sensitivity, detecting about one adduct per 107 bases and ability to screen many samples because of easy automations. The current prerequisite for the assay is that DNA can be modified to sufficiently high levels with the ultimate carcinogen to make it suitably antigenic. These types of antigens have been used to raise polyclonal antibodies in rabbits (41) and monoclonal antibodies from mice (42). 

Generation of antibodies that can recognize and bind to specific viruses is straightforward. A sample of live or attenuated virus, or a purified component of the viral caspid, can be injected into animals to stimulate polyclonal antibody production (or to facilitate monoclonal antibody production by hybridoma technology). Harvested antibodies are then employed to develop specific immunoassays that can be used to screen test samples routinely for the presence of that specific virus. Immunoassays capable of detecting a wide range of viruses are available commercially. The sensitivity, ease, speed and relative inexpensiveness of these assays render them particularly attractive. 

The basic principle for making the rabbit monoclonal antibody is the same as for mouse monoclonals. Rabbit fusion partner cells can fuse to rabbit B-cells to create the rabbit hybridoma cells. Hybridomas are then screened to select for clones with... 

The practical achievement of this goal was held up for 18 years, primarily because of the great difficulty in isolation and purification of single-species proteins from the immune repertoire. During that time, many attempts to elicit catalysis by inhomogeneous (i.e. polyclonal) mixtures of antibodies were made and failed (e.g. Raso and Stollar, 1975 Summers, 1983). The problem was resolved in 1976 by Kbhler and Milstein s development of hybridoma technology, which has made it possible today both to screen rapidly the complete immune repertoire and to produce in vitro relatively large amounts of one specific monoclonal antibody species (Kohler and Milstein, 1975 Kohler et al., 1976). 

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