Development of an immunomagnetic assay system for rapid detection of bacteria and leukocytes in body fluids

The principles of magnetic separation aided by antibodies or other specific binding molecules have been used for isolation of specific viable whole organisms, antigens, or nucleic acids. Whereas growth on selective media may be helpful in isolation of a certain bacterial species, immunomagnetic separation (IMS) technology can isolate strains possessing specific and characteristic surface antigens. Further separation, cultivation, and identification of the isolate can be performed by traditional biochemical, immunologic, or molecular methods. PCR can be used for amplification and identification of genes of diagnostic importance for a target organism. The combination of IMS and PCR reduces the assay time to several hours while increasing both specificity and sensitivity. Use of streptavidin-coated magnetic beads for separation of amplified DNA fragments, containing both biotin and a signal molecule, has allowed for the conversion of the traditional PCR into an easy-to-read microtiter plate format. The bead-bound PCR amplicons can also easily be sequenced in an automated DNA sequencer. The latter technique makes it possible to obtain sequence data of 300 to 600 bases from 20 to 30 strains, starting with clinical samples, within 12 to 24 h. Sequence data can be used for both diagnostic and epidemiologic purposes. IMS has been demonstrated to be a useful method in diagnostic microbiology. Most recent publications describe IMS as a method for enhancing the specificity and sensitivity of other detection systems, such as PCR, and providing considerable savings in time compared with traditional diagnostic systems. The relevance to clinical diagnosis has, however, not yet been fully established for all of these new test principles. In the case of PCR, for example, the presence of specific DNA in a food sample does not demonstrate the presence of a live organism capable of inducing a disease. However, all tests offering increased sensitivity and specificity of detection, combined with reduced time of analysis, have to be seriously evaluated.

The kinetics of antigen-antibody reactions is reviewed with special attention paid to the specific properties at solid-liquid interfaces. Theories of possible diffusion limitation in forward reaction rates are compared to experiments. It is found that the intrinsic forward reaction rate in the bimolecular antigen-antibody reaction is normally not limited by diffusion either in solution or at the solid-liquid interface. However, reactions at the solid-liquid interface can be diffusion limited due to depletion of reactants close to the surface. This effect depends on geometry, intrinsic reaction rate and surface concentration of receptor molecules. Normally cell surface reactions are not diffusion limited whereas reactions at artificial surfaces often are limited by diffusion. When not limited by diffusion it is also found that the intrinsic forward and reverse reaction rates are lower for surface reactions compared to reactions in solution. Antigen-antibody reactions at solid-liquid interfaces can often be considered as practically irreversible and limited by mass transport or steric interactions.

Extremely sensitive detection of various biotoxoids and bacterial spores using the commercial ORIGEN analyzer was achieved by capture on antibody-conjugated micron sized magnetic beads (MBs) followed by binding of ruthenium (II) trisbipyridal chelate (Ru(bpy)2+3-labelled reporter antibodies. Immunomagnetically captured target materials were collected on a magnet. Electrochemiluminescence (ECL) was evoked from the Ru(bpy)3(2+)-tagged reporter antibodies by application of an electrical potential. Femtogram sensitivity levels were obtained for all biotoxoids tested including botulinus A, cholera beta subunit, ricin and staphylococcal enterotoxoid B by this immunomagnetic (IM)-ECL approach. An IM-ECL assay for Bacillus anthracis spores yielded a detection limit of at least 100 spores. The ECL signal was a function of analyte quantity over several orders of magnitude, but the immunological 'hook' effect at high antigen loads made quantitation impossible over a broader range. All assays were performed with a maximum combined incubation and assay time of approximately 40 min. This work demonstrates the extreme sensitivity of the IM-ECL approach for soluble and particulate antigens.