Monoclonal antibodies homogeneous

Monoclonal antibodies Homogeneous antibody populations directed at against a single antigenic determinant, produced by a single clone of cells... 

A monoclonal antibody is a homogeneous serum containing only one antibody directed against an antigen. 

As they differentiate into populations with differing functions, B and T cells acquire molecules on their surfaces that reflect their specializations. It is possible to produce homogenous antibodies of a single specificity, termed monoclonal antibodies, which can recognize such surface markers. When laboratories from all over the world compared the monoclonal antibodies they had raised, it was found that clusters of monoclonal antibodies were recognizing the same molecule on the surface of the lymphocyte. Each surface molecule so defined was referred to as a CD molecule (Table 2), where CD refers to a cluster determinant. 

It should be pointed out that, in contrast with the enzymes of primary metabolism (e.g., photosynthesis, respiration, etc.), those catalyzing the synthesis of secondary metabolites usually occur in very low abundance, and are therefore, very difficult to purify to homogeneity especially for the purpose of raising antibodies. However, the recent advances in immunization and selection techniques made it possible to overcome the need for homogeneous protein in order to raise monoclonal antibodies. 

Factor IX may also be purified by immuno-alfinity chromatography, using immobilized anti-IX murine monoclonals. Purification to homogeneity is particularly important in the case of recombinant products. At least one monoclonal antibody has been raised that specifically binds only to factor IX, which contains pre-bound Ca + (i.e. the Ca +-dependent conformation of factor IX). Immobilization of this antibody allowed the development of an immuno-alfinity system in which factor IX binds to the column in the presence of a Ca -containing bufier. Subsequent elution is promoted simply by inclusion of a chelating agent (e.g. EDTA) in the elution buffer. 

Figure 15.3 T3fpical heterogeneous distribution of binding site affinities found in molecular imprinted polymers compared with the homogenous distribution of a typical monoclonal antibody. Reprinted from Umpleby et al. (2001). Copyright 2001 American Chemical Society.

Catalytic antibodies, like enzymes, must be isolated and purified to homogeneity before they can be studied. Initially this was done by using the hybridoma technique for isolation of monoclonal antibodies (Box 31-A). After induction of antibody formation by injecting a selected hapten into a mouse, large numbers of monoclonal antibodies had to be tested for catalytic activity. Even if several thousand different monoclonal antibodies were tested, only a few with catalytic properties could be found.1 Newer methods have incorporated recombinant DNA techniques (Box 31-A) and use of combinatorial libraries and phage display.) Incorporation of acidic or basic groups into the haptens used to induce antibody formation may yield antibodies capable of mimicking the acid-base catalysis employed by natural enzymes. 0... 

In conclusion, studies on isolated cells allow measurements on homogeneous cell populations under well-defined conditions and can aid in the interpretation of metabolic changes seen by NMR in the same or similar cells in the intact animal. In some cases the cells may be a valid system for study in their own right. For example, there have been several NMR studies of commercially important mammalian cell lines which are used in the biotechnology industry for the production of various monoclonal antibodies and recombinant proteins with therapeutic or analytical applications (Mancuso et al., 1990 Gillies et al., 1991). 

Note that so far the expanded bed mainly has been used with rather uncomplicated systems such as for purification of monoclonal antibodies from culture broth, isolation of extracellular substances from microbial cultivations, harvesting of fusion proteins from Escherichia coli cell homogenates, affinity isolation of certain enzymes from microbial homogenates, and separation of serum proteins from serum. To our knowledge there are very few reports on the isolation from homogenates of mammalian tissues or plant material. 

---