Preparation of antigen for immunization

Preparation of antigens for immunization 5 Conjugation of peptides to carriers 6... 

Fig. 1. Preparation of emulsion for immunization. Two iuer Jock glass syringes connected by a three-way plastic stopcock are used to form a stable emulsion of antigen and adjuvant.

An interesting application of CRIE is the preparative aspect. The separation of proteins into clearly positioned and well-spaced precipitates provides an easy means of obtaining purified antigens for immunization of animals. By loading the gel maximally with a protein sample, there will be sufficient material for successful immunization. 

The simplicity of this assay makes it highly attractive for further development. Some of the limitations include the need for the preparation of relatively large amounts of appropriately modified DNA for the initial immunization. This may be reduced with the development of in vitro immunization techniques in which as little as 5ng of antigen can be effective. The antibodies must also be characterized once prepared. For example, a variety of monoclonal antibodies against B[a]PDE-modified DNA have been prepared (42) which show varying specificity. At one extreme, some require the full structure of the adduct bound to DNA, at the other, BtalP tetraol will effectively compete. What is not clear at the moment is how specific such antisera are for a particular PAH. Will these antisera recognize only B[a]P tetraol structures or those of any diol epoxide modified DNA ... 

Figure 10.3. Schematic representation of monoclonal antibody production using immortalized hybrid cells that secrete antibodies selective for the target antigen. The mortal, immune B cells Isolated from mice immunized with a target antigen are fused with myeloma, immortal B cells that express defective antibodies. The selecting of antigen-specific, immortal hybrid cells (hybridomas) results in identification of unique clones of cells that express antibodies with high specificity and affinity (monoclonal antibodies). These cells are cloned and expanded for large-scale monoclonal antibody preparations.

Apart from the spleen, other lymphoid tissues, such as tonsils and the mesenteric or popliteal lymph nodes, can be used as a source of lymphocytes. In the preparation of MABs of human or veterinary origins it is often not possible to obtain lymphoid tissue, and there have been many reports of the successful use of lymphocytes separated from peripheral blood. In some cases, for ethical or practical reasons, it is not possible to immunize the lymphocyte donor, as when human MABs are required, or acutely toxic antigens are used. Also, antigen is not always available in sufficient quantities to perform a successful immunization in vivo. In these circumstances, it may be possible to perform the boosting stage or, indeed, the entire immunization procedure on the lymphocytes in vitro. 

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