Proteins constitute the majority of antigens in physiology and biotechnology. For puposes of antibody development, proteins can be grouped into several classes: cytoplasmic proteins, nuclear proteins, internal membrane proteins, cell surface receptors, and secreted proteins. Cell surface receptors and secreted proteins are usually glycosylated and referred to as glycoproteins. Immunization strategies vary for the different classes, for example, antibodies against cytoplasmic, nuclear and secreted proteins are usually generated by immunization with purified antigen in the presence of adjuvant. Cell surface receptors are usually not available in quantities sufficient of immunization, unless they are made as recombinant soluble fragments. Alternatively, animals can be immunized with whole cells or cell membranes, spleenocytes fused with myeloma cells, and hybridomas producing the antibody with desired specificity selected by ELISA, flow cytometry, or an applicable functional assay. If antigen is limited but its peptide sequence at least partially known, immunizations can be performed with relatively short synthetic peptides derived from the known sequence. In many instances, the antisera raised against the peptide(s) will recognize the holoprotein in native form and/or on Western Blots. Some proteins are poorly immunogenic. In order to overcome this liability, such proteins or fragments thereof are conjugated to immunogenic carriers and/or used to immunize transgenic animals in which the same antigen or homologue thereof has been deleted.
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