Immunogenicity of a Biological Simulant: Strategies for the Improvement of Antibody-Based Detection

摘要

When biological simulants are used for testing detection/identification instruments, antibodies are often employed to quantitate the amount of simulants or sometimes as an integral part of the detection suite. The bacterium Pantoea agglomerans (formerly Erwinia herbicola, Eh) presently is used to simulate vegetative biological agents, however, anti- Eh antibodies of high affinity and specificity are needed. Therefore, we have characterized rabbit antibodies raised against native Eh and Eh chemically modified with dimitrophenyl groups (Eh-DNP) as a strategy to increase intrinsic Eh antigenicity. Our second strategy is to genetically manipulate Eh cell surface components so that we can employ commercially available antibodies or potentially antibodies to epitopes of known agents displayed on Eh. Here we report progress on characterization of anti-Eh antibodies by ELISA and Western blot analyses, and on expression of foreign genes in Eh in developing the surface display approach. Anti Eh DNP preparations contained antibodies against the DNP label, as expected, and were equally or slightly more efficient in detecting native Eh cells, compared with antibodies raised against native Eh. Affinity purification procedures significantly improved Eh detection sensitivity. Both ELISA and Western blot analyses showed marked cross reactivity against other bacteria such as E. coli. This finding emphasizes the need for an alternative approach for specific detection. We have successfully transformed Eh with several different vectors and have cloned and sequenced a prominent Eh outer membrane protein needed for display of specific polypeptide tags. Ultimately this should extend Eh applicability as a simulants expressing specific markers from selected agents.