Studies of Listeria monocytogenes-antibody binding using electro-orientation

An electro-optical (EO) approach has been used for studies of Listeria monocytogenes–antibody binding. The EO analyzer, which has been developed at the State Research Center for Applied Microbiology, Obolensk, was used as a basic instrument for EO measurements. AC electro-kinetic effects depend on dielectric properties of bioparticles, their composition, morphology, the medium, and the frequency of applied electrical field. Electro-orientational spectra were used for discrimination of bacteria before and after selective binding with antibodies. The measurements were performed using a discrete set of frequencies of the orienting electric field (10, 100, 250, and 500 kHz). During biospecific interactions an antibody is bound to the microorganism causing a change in the dielectric properties of the microorganism–antibody complex and the EO signal reaches its maximum at 100–200 kHz. It has been shown that the biospecific interactions of L. monocytogenes cells with anti-Listeria antibody in the presence of Escherichia coli K-12, and Azospirillum brasilense Sp7 change the EO signals significantly. Thus, the determination of the presence of particular bacteria within a mixed sample may be achieved by selection and matching of antibodies specific to individual bacterium types and by comparing spectra of bacterium in the presence and in the absence of specific antibody.     

Electrooptical properties of the microbial suspensions during a cell’s interaction with the antibodies of a different specificity

The electrooptical abilities of the microbial suspensions during a cells interaction with antibodies (ABs) of a different specificity have been studied on the example of the Azospirillum brasilense Sp245 cells and their interaction with the polyclonal monospecific and polyspecific antibodies. Measuring of the orientational spectra of the cells has been performed using the ELUS electrooptical analyzer. A discrete frequency set of an orienting electric field (740, 1000, 1450, 2000, and 2800 kHz) was used. It has been shown that an interaction of the polyspecific AB with the investigated cells redoubles the value of an electrooptical signal of the cells’ suspension as compared with the monospecific antibodies. These findings can be used for a development a new method of microorganism detection.     

Electrooptical analysis of the Escherichia coli-phage interaction

This article describes electrooptical (EO) characterization of biospecific binding between the bacterium Escherichia coli XL-1 and the phage M13K07. The electrooptical analyzer (ELUS EO), which has been developed at the State Research Center for Applied Microbiology, Obolensk, Russia, was used as the basic instrument for EO measurements. The operating principle of the analyzer is based on the polarizability of microorganisms, which depends strongly on their composition, morphology, and phenotype. The principle of analysis of the interaction of E. coli with the phage M13K07 is based on registration of changes of optical parameters of bacterial suspensions. The phage-cell interaction includes the following stages: phage adsorption on the cell surface, entry of viral DNA into the bacterial cell, amplification of phage within infected host, and phage ejection from the cell. In this work, we used M13K07, a filamentous phage of the family Inoviridae. Preliminary study had shown that combination of the EO approach with a phage as a recognition element has an excellent potential for mediator-less detection of phage-bacteria complex formation. The interaction of E. coli with phage M13K07 induces a strong and specific EO signal as a result of substantial changes of the EO properties of the E. coli XL-1 suspension infected by the phage M13K07. The signal was specific in the presence of foreign microflora (E. coli K-12 and Azospirillum brasilense Sp7). Integration of the EO approach with a phage has the following advantages: (1) bacteria from biological samples need not be purified, (2) the infection of phage to bacteria is specific, (3) exogenous substrates and mediators are not required for detection, and (4) it is suitable for any phage-bacterium system when bacteria-specific phages are available.     

Changes in electrophysical characteristics of Azospirillum brasilense Sp7 cells during their interaction with polyclonal antibodies

Electrooptical characteristics of Azospirillum brasilense Sp7 cells during their specific interaction with polyclonal rabbit antibodies were studied. Dependence of optical density of cell suspension during electroorientation of cells from frequency of orienting field in interval 10, 100, 250, and 500 kHz was evaluated. Itwas shown that electrooptical (EO) characteristics of bacterial suspensions change during interaction of A. brasilense cells with antibodies, and maximal changes occur when frequency of oriented field amounts 100-250 kHz. During interaction of A. brasilense Sp7 with strain-specific polyclonal antibodies in the presence of Escherichia coli K-12 and Pseudomonas putida C-11 decrease of amplitude of analytic signal was observed but detection of A. brasilense Sp7 cells was possible. Possibility of detection of microorganisms by EO analysis during their interaction with antibodies was shown.     

Composite surface for blocking bacterial adsorption on protein biochips

The design and fabrication of protein biochips requires characterization of blocking agents that minimize nonspecific binding of proteins or organisms. Nonspecific adsorption of Escherichia coli, Listeria innocua, and Listeria monocytogenes is prevented by bovine serum albumin (BSA) or biotinylated BSA adsorbed on SiO(2) surfaces of a biochip that had been modified with a C(18) coating. Biotinylated BSA forms a protein-based surface that in turn binds streptavidin. Because streptavidin has multiple binding sites for biotin, it in turn anchors other biotinylated proteins, including antibodies. Hence, biotinylated BSA simultaneously serves as a blocking agent and a foundation for binding an interfacing protein, avidin or streptavidin, which in turns anchors biotinylated antibody. In our case, the antibody is C11E9, an IgG-type antibody that binds Listeria spp. Nonspecific adsorption of another bacterium, Escherichia coli, is also minimized due to the blocking action of the BSA. The blocking characteristics of BSA adsorbed on C(18)-derivatized SiO(2) surfaces for construction of a protein biochip for electronic detection of pathogenic organisms is investigated.     

Electrooptical properties of soil nitrogen-fixing bacterium Azospirillum brasilense: effect of copper ions

The effects of copper ions on the uptake of some essential metals in the biomass and the electrooptical properties of cell suspensions of the nitrogen-fixing soil bacterium Azospirillum brasilense sp. 245 were studied. Copper cations were shown to be effectively taken up by the cell biomass from the culture medium. The addition of copper ions increased the rate of uptake of some other metals present in the culture medium. This was accompanied by changes in the electrooptical characteristics of cell suspension as measured within the orienting electric field frequency range of 10 to 10,000 kHz. The effects observed during short-term incubation of A. brasilense in the presence of copper cations were less significant than during long-term incubation. These results can be used for rapid screening of microbial cultures for enhanced efficiency of sorption and uptake of metals.     

Electrophysical characteristics of Azospirillum brasilense Sp245 during interaction with antibodies to various cell surface epitopes

This work was undertaken to examine the electrooptical characteristics of cells of Azospirillum brasilense Sp245 during their interaction with antibodies developed to various cell surface epitopes. We used the dependences of the cell suspension optical density changes induced by electroorientation on the orienting field frequency (740, 1000, 1450, 2000, and 2800kHz). Cell interactions with homologous strain-specific antibodies to the A. brasilense Sp245 O antigen and with homologous antibodies to whole bacterial cells brought about considerable changes in the electrooptical properties of the bacterial suspension. When genus-specific antibodies to the flagellin of the Azospirillum sheathed flagellum and antibodies to the serologically distinct O antigen of A. brasilense Sp7 were included in the A. brasilense Sp245 suspension, the changes caused in the electrooptical signal were slight and had values close to those for the above changes. These findings agree well with the immunochemical characteristics of the Azospirillum O antigens and with the data on the topographical distribution of the Azospirillum major cell surface antigens. The obtained results can serve as a basis for the development of a rapid test for the intraspecies detection of microorganisms.     

Comparison of techniques to screen and characterize bacteria-specific hybridomas for high-quality monoclonal antibodies selection

Antibodies are very important materials for diagnostics. A rapid and simple hybridoma screening method will help in delivering specific monoclonal antibodies. In this study, we systematically developed the first antibody array to screen for bacteria-specific monoclonal antibodies using Listeria monocytogenes as a bacteria model. The antibody array was developed to expedite the hybridoma screening process by printing hybridoma supernatants on a glass slide coated with an antigen of interest. This screening method is based on the binding ability of supernatants to the coated antigen. The bound supernatants were detected by a fluorescently labeled anti-mouse immunoglobulin. Conditions (slide types, coating, spotting, and blocking buffers) for antibody array construction were optimized. To demonstrate its usefulness, antibody array was used to screen a sample set of 96 hybridoma supernatants in comparison to ELISA. Most of the positive results identified by ELISA and antibody array methods were in agreement except for those with low signals that were undetectable by antibody array. Hybridoma supernatants were further characterized with surface plasmon resonance to obtain additional data on the characteristics of each selected clone. While the antibody array was slightly less sensitive than ELISA, a much faster and lower cost procedure to screen clones against multiple antigens has been demonstrated.     

Electrooptical Properties of Cells of the Soil Nitrogen-Fixing Bacterium Azospirillum brasilense: Effects of Copper Ions

The effects of copper ions on the uptake of some essential metals in the biomass and the electrooptical properties of cell suspensions of the nitrogen-fixing soil bacterium Azospirillum brasilensesp. 245 were studied. Copper cations were shown to be effectively taken up by the cell biomass from the culture medium. The addition of copper ions increased the rate of uptake of some other metals present in the culture medium. This was accompanied by changes in the electrooptical characteristics of cell suspension as measured within the orienting electric field frequency range of 10 to 10000 kHz. The effects observed during short-term incubation of A. brasilensein the presence of copper cations were less significant than during long-term incubation. These results can be used for the rapid screening of microbial cultures for the enhanced efficiency of sorption and the uptake of metals.     

Isolation and PCR amplification of a species-specific oxidoreductase-coding gene region in Listeria grayi

Listeria grayi is a nonpathogenic Gram-positive bacterium that demonstrates considerable similarities to other members in the genus Listeria, including the foodborne human pathogen Listeria monocytogenes and the animal pathogen Listeria ivanovii. A rapid diagnostic test to identify and diagnose listeriosis would be valuable, especially in cases where the presence of L. grayi may complicate diagnosis. This test would be based on a unique gene present in L. grayi. In this study, after comparative screening of a recombinant L. grayi DNA library by dot blot hybridization, an L. grayi specific clone (lgr20-246) with an insert of 722 bp was isolated. By applying PCR primers derived from a distinct region of the clone not shared by other bacteria, a specific band of 420 bp was amplified from the genomic DNA of L. grayi only and not of other Listeria species or common bacteria. These results suggest that the PCR assay employing primers lgr20-246F and lgr20-246R provides an independent and precise means of distinguishing L. grayi from other Listeria species and common bacteria. Therefore, it would be another useful technique for laboratory differentiation of Listeria bacteria.     

Complement deposition by antibodies to Pseudomonas aeruginosa mucoid exopolysaccharide (MEP) and by non-MEP specific opsonins.

The failure of cystic fibrosis patients to limit chronic infection due to mucoid Pseudomonas aeruginosa might be due to ineffective opsonins produced against this bacterium. Nonopsonizing antibody to the bacterial capsule, mucoid exopolysaccharide (MEP), appears at elevated titers during chronic colonization of cystic fibrosis patients, as do opsonins not specific for MEP. Nonopsonic antibodies to MEP occur naturally in most adults and can be induced in animals by immunization. A limited number of humans produce MEP-specific opsonic antibodies after immunization. The purpose of this study was to compare the activation and deposition of C components onto the bacterial surface in the presence of these different antibodies. Opsonic killing uses the classical C pathway. MEP-specific opsonic and nonopsonic antibodies bound to whole bacteria and activated C to a comparable degree, but opsonic antibody deposited 3 to 40 times more C3 onto bacteria, mostly as C3bi, compared to nonopsonic antibody. In addition, two to three times as much nonopsonic mAb as opsonic mAb (both IgG2b) bound to the bacteria at comparable input concentrations, indicating the difference in C deposition was not due to differences in antibody binding. Non-MEP-specific opsonins also bound C3 to the bacteria, but only a mean of 27 +/- 14% was ester linked, compared with 81 +/- 11% of C3 deposited by MEP-specific opsonins. Immunoprecipitation experiments indicated that two-thirds of the C3 bound in the presence of MEP-specific opsonins was linked to MEP, whereas non-MEP-specific opsonins obtained from infected patients deposited the C3 onto LPS and other unidentified Ag. These data show that MEP-specific opsonins function by depositing C3 onto the outer bacterial surface that differentiates them from non-MEP-specific opsonins produced in response to chronic infection.     

Comparison of the electrooptical properties and specific respiratory activity of Acinetobacter calcoaceticum A-122

The electrooptical (EO) properties of a cell suspension and the specific respiratory activity of cells towards p-nitrophenol (PNP) were compared during PNP metabolism in Acinetobacter calcoaceticum strain A-122. The frequency dependence of the suspension's turbidity changes due to cellular orientation (orientational spectra) at frequencies of an orienting electric field of 10-10,000 kHz was determined. Orientational spectral changes observed during PNP incubation of the cells were followed over the range of 10-502 kHz. There were linear relationships between the magnitude of the EO effect at a 502-kHz frequency and the concentration of PNP over the range of 0.1-0.8 mM, and between the specific respiratory activity of the cells and the concentration of PNP over the range of 0.1-1.0 mM. The knowledge gained from these studies suggests a direct relationship between alterations in the cellular EO properties and PNP metabolism.     

Exploitation of the electro-optical characteristics of microbial suspensions for determining p-nitrophenol and microbial degradative activity

The effect of p-nitrophenol metabolism on the electrophysical properties of a microbial cell suspension of Acinetobacter calcoaceticum was investigated. The dependence of the suspension's optical density changes due to the orientation of the electric field on the frequency of an orienting electric field (orientational spectra) over the range 10–10 000 kHz was used. Upon optimization of cell culture conditions, followed by orientational spectrum measurements, it was found that the most dramatic changes in orientation spectrum associated with p-nitrophenol metabolism took place over the frequency range 10–1000 kHz. Additionally, a linear dependence of the magnitude of the electrooptical effect on p-nitrophenol concentrations of 0.1–0.8 mM was established. The minimum p-nitrophenol concentration detectable by this calibration was 0.1 mM.     

A smart membrane based on an antigen-responsive hydrogel

Hydrogel membranes have been fabricated that incorporate antibody/antigen moieties. The permeability of large solutes through these membranes is dependent on the presence of soluble antigen that can compete with the internal interactions between antibody and antigen leading to an increase in gel mesh size. Specifically, the membrane's structure is based on a dextran backbone grafted with a fluorescein isothiocyanate (FITC) antigen and a sheep anti-FITC IgG antibody. The backbone is covalently cross-linked by conjugated divinyl sulfone (DVS) groups. The gel structure is additionally stabilized by affinity crosslinks formed by biospecific interactions between the bound IgG and FITC. FTIR spectra of the gel are consistent with formation of covalent bonds between cysteine groups in the IgG and DVS groups in the dextran. Results obtained using isothermal titration calorimetry (ITC) confirmed the competitive interaction binding between IgG-FITC-dextran and free sodium fluorescein at pH 5.0. Scanning electron microscopy (SEM) of samples prepared using cryofixation and cryofracturing techniques showed that observed changes in permeability correlate with free fluorescein-dependent structural changes in the gel. Three-dimensional images obtained from confocal laser scanning microscopy show that these changes occur throughout the gel and indicate that SEM results are not artifacts of sample preparation. The permeability of these gels, as shown by blue-dextran (12 kDa) diffusion, increases in response to the presence of free fluorescein of the external medium, which causes competitive displacement of the affinity cross-links. Sequential addition and removal of sodium fluorescein showed that these permeability changes are reversible.     

Multifunctional inorganic-binding beads self-assembled inside engineered bacteria

Multifunctional shell-core nano/microbeads with a hydrophobic biopolymer core and a designed protein coat for selective binding of an inorganic substance and antibodies were self-assembled inside engineered bacteria. Hybrid genes were constructed to produce tailormade bead-coating proteins in the bacterium Escherichia coli. These fusion proteins contained a binding peptide for an inorganic material, the antibody binding ZZ domain, and a self-assembly promoting as well as biopolymer synthesizing enzyme. Production of these multidomain fusion proteins inside E. coli resulted in self-assembly of beads comprising a biopolyester core and displaying covalently bound binding sites for specific and selective binding of an inorganic substance and any antibody belonging to the immunoglobulin G class. Engineered beads were isolated and purified from the respective E. coli cells by standard cell disruption procedures. Bead morphology and the binding functionalities displayed at the bead surface were assessed by the enzyme-linked immunosorbent assay, transmission electron microscopy, elemental analysis, backscattering electron density, analytical density ultracentrifugation, and atomic force microscopy. These analyses showed that bacteria can be engineered to produce fusion proteins mediating self-assembly of spherical biopolymer beads with binding affinity to gold and/or silica and antibodies. Spherical structures of this type could conceivably serve as nano/microdevices for bioimaging in medical approaches where an antibody mediated targeted delivery of an inorganic contrast agent would be desired.     

Changes in electrophysical properties of Escherichia coli K-12 cells under the action of kanamycin and tetracycline

Essential changes in the orientation spectra (OS) of cell suspensions incubated at different concentrations of kanamycin, were found to occur only in the first 5 frequencies of the orienting electrical field (10-1,000 kHz)). The maximum change in the intensity of the electrooptical signal occurred at a concentration of kanamycin of 10 microg/ml. Antibiotic concentration of 5 microg/ml caused no changes in OS. During the incubation of the cells with tetracycline (1.7, 2.5, 5.0 microg/ml) no changes in OS of the cell suspension were registered. Considerable changes in the intensity of the electrooptical signal occurred during the incubation of the cells with kanamycin (5 microg/ml) and tetracycline (1.7 microg/ml) simultaneously, which was due to the synergic action of these two antibiotics. Thus, as found with the use of the electrooptical analysis, the joint action of kanamycin and tetracycline could increase their antibacterial effect. The results demonstrated the effectiveness of using electrophysical methods for the study of the mechanism of action of antibiotics on bacterial cells and for the control of such action.     

Traditional ELISA methods for antibody affinity determination fail to reveal the presence of low affinity antibodies in antisera: an alternative approach

Traditionally used methods of antibody affinity determination either by ELISA or by the surface plasmon resonance technique do not allow detection of the presence of low‐affinity antibodies in samples of high‐affinity antibodies. In this paper we demonstrate the possibility to reveal their presence and to determine the affinities of both categories of antibodies as well as the ratio of their concentrations. This is especially important since by using traditional methods for antibody affinity evaluation the admixture of low‐affinity antibodies in a sample diminishes the accuracy in determination of specific antibody affinity. In addition, the presence of an admixture of low‐affinity antibodies may be an important biological characteristic of the system under study; their revelation and the evaluation of their binding parameters may be valuable in many cases for obtaining a more complete characterization of the binding properties of the multiple antibodies generated in an immune response. Copyright © 2009 John Wiley & Sons, Ltd.     

The effect of ampicillin on the electrophysical properties of Escherichia coli cells

The study of the effect of ampicillin on the electrophysical properties of Escherichia coli cells showed that this antibiotic influences the orientational spectra (OS) of the ampicillin-susceptible E. coli strains K-12 and XL-1 within the frequency range 10-1000 kHz of the orienting electric field and does not affect the OS of the ampicillin-resistant strains K-12(pUC-18) and XL-1(pHEN1). The change in the electrooptical signal of the ampicillin-susceptible cells was maximum at an ampicillin concentration of 50 microg/ml and did not depend on the exposure time. The conclusion is drawn that changes in the OS of cells can be used to evaluate their resistance to ampicillin.     

Circular dichroism and fluorescence studies of homogeneous antibodies to type III pneumococcal polysaccharide.

The near-ultraviolet circular dichroism (CD) of three homogeneous anti-type III pneumococcal antibodies in the absence and the presence of the specific hexasaccharide ligand was studied. In addition recombinations and hybridizations of H and L chains derived from two of these antibodies were carried out and the CD spectra of bound and free reconstituted IgG molecules were measured. The results indicate that the CD spectra of the native antibodies in the 260-310-nm range are very similar in shape and sign and exhibit a positive band at 285 nm. The homologous reconstituted antibody molecules exhibited CD spectra very similar in shape and sign to those of the native antibody molecules although recombinant molecules are no longer stabilized by interchain disulfide bonds. Upon addition of the hexasaccharide ligand, a significant decrease in amplitude of the CD spectra (18-21%) occurred in all three native antibodies and their Fab fragments as well as in the homologous recombinant molecules. No CD spectral changes could be detected upon interaction of the hapten ligand with the heterologous recombinants. All homogeneous antibodies studied exhibited fluorescence quenching upon oligosaccharide binding and a blue shift of the emission maximum. This property allowed the determination of the binding constant of one selected antibody to be made. Taken together, CD and fluorescence spectroscopic data suggest that oligosaccharide ligands induced detectable conformational changes in the Fab fragment of the antibody.     

Electrooptical parameters of kanamycin-treated E. coli cell suspensions

The effect of kanamycin on the electrophysical parameters of cell suspensions of Escherichia coli K-12 and pMMB33 was investigated. Incubation of the sensitive K-12 strain with kanamycin resulted in significant changes in the orientation spectra (OS) of the cell suspensions; these changes were not revealed in the case of the resistant pMMB33 strain. In the case of the sensitive K-12 strain incubated with different kanamycin concentrations, changes in the OS of the cell suspensions occurred within the 10-1000 kHz frequency range of the orienting electrical field. The most pronounced change in the electrooptical signal was observed at 10 microg/ml of kanamycin. Control experiments were carried out by standard plating on nutrient media. Thus, the OS changes of suspensions in the presence of antibiotics may be used as a test for microbial resistance to such antibiotics.