A New Approach to Salmonella Detection and Serogroup Differentiation Using Murine Monoclonal Antibodies

This dissertation, "A New Approach to Salmonella Detection and Serogroup Differentiation Using Murine Monoclonal Antibodies" by 吳子柏, Sze-park, Ng, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract With two newly raised murine monoclonal antibodies, MO7.1 and MO 15, in addition to a panel of seven others, two immune capture enzyme-linked immunosorbent assays were developed. One is intended for detection, and the other, for serogroup differentiation, of Salmonella. The assays make use of microtiter plate previously coated with a pan Salmonella monoclonal antibody (T6) to selectively bind Salmonella from test samples. The bound Salmonella are detected using an enzyme conjugate of T6, or differentiated in serogroups using a panel of seven enzyme conjugated monoclonal antibodies specific for major O antigens of Salmonella serogroups A, B, Q, 2, DI and E. The results show that the assays were capable of sensitive and specific detection of over 95% of Salmonella affecting man and afforded reliable differentiation of major Salmonella serogroups. These assays facilitate identification and differentiation of an important group of human pathogens and have found useful applications in food and environmental microbiology. The antigenic specificity of the antibodies used in these assays was analysed by comparing O antigen structures of Salmonella and control gram-negative bacterial strains. The immunodominant disaccharide recognized by pan Salmonella antibody, T6, was most probably the terminal N-acetylglucosamine-glucose residue located in the outer core polysaccharide which is conserved among Salmonella." Differentiation of serogroup A, B and DI, was effected using the O factor specific antibodies, MO2, MO4 and MO9, respectively. The immunodominant sugars identified by these antibodies were most probably optical isomers of di-deoxy-hexose residues, paratose, abequose and tyvelose, respectively, since O antigen structures of these serogroups are otherwise identical. Serogroup Ca was differentiated using MO8. The immunodominant sugar recognized by this antibody was most probablyabequose, as in serogroup B, but the residue was recognized by MO8 in association with a different O antigen structure, such that the antibody was not also reactive with serogroup B, nor MO4 with serogroup Ci. MO7 reacted exclusive with non- lysogenic serogroup Cl strains with the third mannose on the backbone playing a dominant role. MO7.1, a newly raised monoclonal antibody, specifically reacts with both lysogenic and non-lysogenic strains of serogroup Q. Its specificity was attributed to the N-acetylglucosamine and probably the first mannose residue on the backbone LPS. It was shown that relocation of the glucose residue from the third to another mannose residue due to phage 14 lysogeny abrogated MO7's reactivity but not that of MO7.1's. The specificity of MOID was mainly attributed to alpha di-ester linkage between repeated oligosaccharides making up O antigen of serogroup El Salmonella, whereas another new monoclonal antibody, MO 15, mainly recognizes beta di-ester linkage of an otherwise identical backbone LPS structure of serogroups ET and E salmonella. All O factor specific antibodies used in this study also effected specific and efficient agglutination of the corresponding serogroups. This presumably was due to repeated representation of the respective antigenic epitopes on O antigens. The bacterial agglutination assay, unlike the immune capture assays described above, were carried out using heavy bacterial suspension and thus under conditions of high- abundance of anti