A new approach to Salmonella detection and serogroup differentiation using murine monoclonal antibodies
With two newly raised murine monoclonal antibodies, M07.1 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 microliter 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, Cz, 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, M02, M04 and M09, 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 C2 was differentiated using MO8. The immunodominant sugar recognized by this antibody was most probably
abequose, as in serogroup B, but the residue was recognized by M08 in association with a different O antigen structure, such that the antibody was not also reactive with serogroup B, nor M04 with serogroup C2. M07 reacted exclusive with non-lysogenic serogroup CI strains with the third mannose on the backbone playing a dominant role. M07.1, a newly raised monoclonal antibody, specifically reacts with both lysogenic and non-lysogenic strains of serogroup Ci. 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 M07's reactivity but not that of M07.1's. The specificity of MO10 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 E3 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 antigen. Whereas the immune capture assay favours detection of dominant antigenic determinants, therefore, agglutination assay additionally detected minor antigenic determinants present in the test bacterial populations. Thus, apart from serogroup B, M04 was found to additionally agglutinate serogroup A Salmonella and whereas M02 was reactive mainly with serogroup A, it also agglutinated some strains of serogroup Di organisms. Such apparent cross reactivity
observed under conditions of antigen excess was attributed to the possible presence of biosynthesis intermediates in the epimerization of di-deoxysugars. Under similar assay conditions of antigen excess, there was an increased cross-reaction of M07 against serogroup C4 and an increased cross-reaction of MO 10 against the lysogenic E4 strain. This was probably attributed to incomplete glycosylation of the respective O antigens.
The results described above served to highlight the exquisite specificity of monoclonal antibodies. The structural features recognized by these antibodies, such as those mentioned above, could not be readily studied by conventional means, and this had posed a significant obstacle in the detailed study of antigenic structures of Salmonella O antigens. Therefore, availability of well-defined monoclonal antibodies, such as those presently described, is expected to facilitate future research in this area. Their defined specificity and ready availability also represent a significant advantage over O-factor specific hyperimmune antisera used in the past for identification and differentiation of this important group of human pathogens. Indeed, comparison of results obtained by these two types of reagents showed that monoclonal antibodies can overcome some of the difficulties encountered previously using O-factor specific antisera. Nevertheless, it was shown that serogroup differentiation using monoclonal antibodies was essentially similar to that effected by. conventional O factor specific antisera. This confirmed that the basic Kauffmann and White Scheme laid down more than 50 years ago based on ill defined reagents remain sound to this day. The correlation between results obtained by the two types of reagents provides a rational ground to replace the conventional antisera with monoclonal antibodies for identification and differentiation of Salmonella in the future.
School:The University of Hong Kong
School Location:China - Hong Kong SAR
Source Type:Master's Thesis
Keywords:salmonella monoclonal antibodies
Date of Publication:01/01/1999