Immunoassays with chemically modified bacteriophage

Master Thesis


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University of Cape Town

The immunospecific inactivation of bacteriophage is one of the most sensitive methods available for the detection of very low concentrations of antibody. By chemically modifying the phage coat-protein, this sensitivity can be extended to antibodies against a wide variety of haptens and proteins. Phage particles that have been modified by attaching some molecule onto their surface are sensitive to antibodies directed against the coupled chemical moiety. Furthermore, the inactivation of the modified phage by antibody can be inhibited by free antigen, and this provides a sensitive assay for small quantities of antigen. Antibody and antigens have been detected at the nanogram level by this technique. The modified phage technique can also be used to distinguish antibodies of different specificities and to discriminate between closely related antigens. This technique has not yet been applied to the immunochemical study of viral components and the present work represents such an attempt. Tobacco mosaic virus (TMV) was chosen as a model system since it permits the study of numerous inununological phenomena (Rappaport, 1965; van Regenmortel, 1966). A series of preliminary experiments were performed to obtain experience in the methodology of the technique. These included the inactivation of native T4 phage by phage antiserum and anti phage IgG, the chemical modification of phage with DNP and the attachment of lysozyme to phage under a variety of conditions. The success of the covalent binding of protein to phage particles depends on finding conditions under which a proportion of the phage remains viable and, at the same time, can still be neutralized by anti-protein sera. To this end, different proportions of reactants and three different bifunctional reagents were tested. To prevent aggregation of tobacco mosaic virus protein (TMVP) at the high concentration used, the protein was treated with N-bromosuccinimide. TMVP-phage conjugates which were sensitive to antiserum were prepared using bis-diazobenzidine as the bifunctional reagent.

Bibliography: pages 163-176.