Improved Detection Sensitivity of a Biological Simulant: Delivery of Chemical Labels to the Cell Surface by Adsorption of Polyethyleneimine Derivatives

摘要

The use of biological simulants in testing detection/identification instruments often employs antibodies to quantify the amount of simulants or sometimes as an integral part of the detection suite. At present, the bacterium Pantoea agglomerans (formerly Erwinia herbicola, Eh) is an approved vegetative agent simulant, however, Eh detection sensitivity is disappointing in standard methods such as ELISA, using various anti-Eh antibody preparations, including antibodies that have been raised against surface- modified Eh in attempts to increase Eh immunogenicity. In addition, anti-Eh antibodies exhibit low specificity for Eh, and react almost as strongly with other enterobacterial species, e.g., E. coti, limiting their value for identification purposes. Therefore, we are pursuing two alternative strategies, genetic manipulation of Eh cell surface components, and chemical modification of the negatively charged surface of Eh to introduce specific groups for detection with antibodies and/or with other highly specific and sensitive tests already available. Here we report progress on the development of a method to deliver onto the surface of Eh large numbers of small molecule labels multiply attached to a polycationic polyethyleneimine (PEI) carrier by strong electrostatic adsorption of the derivatized polymers to cells. Biotin (Bio) or dinitrophenyl (DNP) groups were covalently linked to PEI of average MW 60,000, bearing approx. 1,400 positive charges, by reaction of 1% w/v PEI with N-hydroxysuccinimide-biotin (NHS- biotin) or dinitrobenzenesulfonic acid (DNBS), respectively. Limiting amounts of NHS-biotin and DNBS reagents were employed to incorporate either approximately 50 or approximately 100 Bio or DNP groups per molecule of PEI, leaving a majority of positively charged groups unaltered.