Monoclonal antibodies to Escherichia coli 50S ribosomes.

Hybridoma cell lines that produce monoclonal antibodies directed against 50S Ribosomal proteins have been isolated. Spleen cells (from BALB/c mice immunized with 50S ribosomal subunits extracted from Escherichia coli) were fused to mouse myeloma cell line SP2/O-Ag 14. The initial screening for antibody producing hybridomas was carried out by a double antibody sandwich method; hybridomas were subsequently cloned in soft agar. Antibodies were characterized by their specific binding to individual 50S ribsomal proteins separated on phosphocellulose columns and in two-dimensional polyacrylamide gels. The assignments were confirmed with purified single ribosomal proteins. Of four clones analyzed thus far, two are identical with specificity for r-protein L5. The other clones produce two different antibodies directed against r-protein L20. Each monoclonal antibody formed ribosome dimers visualizable in the electron microscope. Dimers could be reacted with a different second antibody to form chains containing 8 or more ribosomes, which may be useful for structural studies.     

Serotype-specific monoclonal antibodies against the H12 flagellar antigen of Escherichia coli

The flagellar filaments of morphotype E isolates of Escherichia coli characteristically possess an apparent helically arranged sheath structure, surrounding the central core of the filament. Re-examination of the type strains of H-serotypes belonging to morphotype E showed that all but serotype H34 possessed the expected morphology. Heterogeneity was observed in both the diameter of filaments from individual morphotype E strains and in the Mr of individual flagellins. There was no apparent correlation between these two features. Monoclonal antibodies (MAbs) of the IgM class were raised against serotype H12 flagella. In Western immunoblotting and agglutination tests, the MAbs recognized the H12 antigen of six isolates with different O:K antigen combinations. The MAbs were H-serotype-specific, with no significant reaction with the H-antigens of other morphotype E strains. The location of the serotype-specific H12 epitope(s) was studied by immunolabelling with colloidal gold markers. The epitope was surface-exposed and appeared to be helically arranged on the flagellar filament. The pattern of colloidal gold labelling was consistent with the possibility that the H12 serotype-specific epitope resides in the apparent sheath structure.     

Monoclonal antibodies for the structural analysis of the Na+/H+ antiporter NhaA from Escherichia coli

Since their advent some 25 years ago, monoclonal antibodies have developed into powerful tools for structural and functional analysis of their cognate antigens. Together with the respective antigen binding fragments, antibodies offer exclusive capacities in detection, characterization, purification and functional assays for every given ligand. Antibody-fragment mediated crystallization represents a major advance in determining the three-dimensional structure of membrane-bound protein complexes. In this review, we focus on the methods used to generate monoclonal antibodies against the NhaA antiporter from Escherichia coli as a paradigm of secondary transporters. We describe examples on how antibodies are helpful in understanding structure and function relationships for this important class of integral membrane proteins. The generated conformation-specific antibody fragments are highly valuable reagents for co-crystallization attempts and structure determination of the antiporter.     

Monoclonal antibodies inhibiting RNA polymerase from Escherichia coli

Monoclonal hybridoma antibodies directed against RNA polymerase from E. coli have been obtained. Only a few have been found to inhibit the enzyme activity. Antibodies produced by two clones, PYN-1 and PYN-2, inhibit RNA polymerase at the stage of RNA chain elongation. The PYN-1 antibodies react with the beta'-subunit of the enzyme. The PYN-2 antibodies react with the beta-subunit and with its 130 kDa amber fragment.     

Immunoliposome sandwich assay for the detection of Escherichia coli O157:H7

We describe the development of a field-portable colorimetric immunoassay for the detection of Escherichia coli O157:H7, using antibody-directed liposomes (immunoliposomes) encapsulating dye as an analytical reagent. Antibodies (anti-E. coli O157:H7) thiolated by 2-iminothiolane were coupled to malemide-tagged liposomes encapsulating the marker dye, sulforhodamine B. Transmission electron microscopy showed that the immunoliposomes bound only to the serotype without any cross-reactivity with tested negative controls. A wicking reagent containing immunoliposomes and the test sample and a plastic-backed nitrocellulose strip with a measurement zone were used in a sandwich (noncompetitive) assay format. During the capillary migration of the wicking reagent, E. coli, with surface-bound immunoliposomes, was captured at the measurement zone on which antibodies to E. coli O157:H7 were immobilized. The color density of the measurement zone was directly proportional to the amount of E. coli O157:H7 in the sample. The detection limit of the current assay with pure cultures of the serotype was ca. 10(4) colony-forming units (CFU)/mL. The assay, which does not need washing and incubation steps, can be completed in 8 min. These results demonstrate the feasibility of using dye-encapsulating immunoliposomes in microporous membranes for the rapid detection of molecules with multivalent antigenic sites.     

Monoclonal antibodies against Stx1B subunit of Escherichia coli O157:H7 distinguish the bacterium from other bacteria

Aims: The Shiga‐like toxins (Stx) are critical virulence factors of enterohaemorrhagic Escherichia coli (EHEC). Stx1B subunit plays important roles in EHEC infection. This work aims to generate and characterize monoclonal antibodies (mAbs) against the Stx1B and to investigate their utility in discrimination ELISA. Methods and Results: Two newly identified mAbs (designated 2H8 and 1B10, respectively) against the Stx1B protein were prepared via hybridoma techniques. The immunoreactivity of both mAbs to the Stx1B protein was confirmed in ELISA and Western blot. Moreover, they differentiate EHEC from Salmonella enteritis, non‐Stx1‐producing E. coli, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus agalactiae and Staphylococcus aureus. Conclusions: The anti‐STx1B mAbs are valuable diagnostic reagents for distinguishing EHEC from other bacteria. Significance and Impact of the Study: This is the first report regarding the usage of anti‐STx1B mAbs in discrimination ELISA. The established ELISA may have potential in clinical surveillance of EHEC infection.     

Liposome-based microcapillary immunosensor for detection of Escherichia coli O157:H7

Our group has previously reported a sandwich-based strip immunoassay for rapid detection of Escherichia coli O157:H7 [Anal. Chem. 75 (2003) 4330]. In the present study, a microcapillary flow injection liposome immunoanalysis (mFILIA) system was developed for the detection of heat-killed E. coli O157:H7. A fused-silica microcapillary with anti-E. coli O157:H7 antibodies chemically immobilized on the internal surface via protein A served as an immunoreactor/immunoseparator for the mFILIA system. Liposomes tagged with anti-E. coli O157:H7 and encapsulating a fluorescent dye were used as the detectable label. In the presence of E. coli O157:H7, sandwich complexes were formed between the immobilized antibodies in the column, the sample of E. coli O157:H7 and the antibody-tagged sulforhodamine-dye-loaded liposomes. Signals generated by lysing the bound liposomes with 30 mM n-octyl-beta-D-glucopyranoside were measured by a fluorometer. The detected signal was directly proportional to the amount of E. coli O157:H7 in the test sample. The mFILIA system successfully detected as low as 360 cells/mL (equivalent to 53 heat-killed bacteria in the 150 microL of the sample solution injected). MeOH (30%) was used for the regeneration of antibody binding sites in the capillary after each measurement, which allowed the immunoreactor/immunoseparator to be used for at least 50 repeated assays. The calibration curve for heat-killed E. coli O157:H7 has a working range of 6 x 10(3)-6 x 10(7)cells, and the total assay time was less than 45 min. A coefficient of variation for triplicate measurements was < or =8.9%, which indicates an acceptable level of reproducibility for this newly developed method.     

Direct PCR detection of Escherichia coli O157:H7

AIMS: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC). METHODS AND RESULTS: Direct PCR (DPCR) obviates the need for the recovery of cells from the sample or DNA extraction prior to PCR. Primers specific for Stx-encoding genes stx1 and stx2 were used in DPCR for the detection of E. coli O157:H7 added to environmental water samples and milk. CONCLUSIONS: PCR reactions containing one cell yielded a DPCR product. SIGNIFICANCE AND IMPACT OF THE STUDY: This should provide an improved method to assess contamination of environmental and other samples by STEC and other pathogens.     

Multiplex PCR for detection of Escherichia coli O157:H7 in foods

Escherichia coli O157:H7 is well known enterohemorrhagic pathogen responsible for infections among animals including a man. The main source of this bacterium is cattle, that is mostly asymptomatic and through that E. coli O157:H7 can simple transfer to food products. Therefore, there is a need for rapid, sensitive and specific detection method. The present work is focused on its detection by a heptaplex polymerase chain reaction, which targets genes from known virulent regions of E. coli O157:H7. According to obtained results this approach is able to reach the detection sensitivity of 4 colony-forming units (CFU) from a culture and 6 and 8 CFU from milk and meat samples, respectively, independently of tested sample volume.     

DNA microdevice for electrochemical detection of Escherichia coli 0157:H7 molecular markers

An electrochemical DNA sensor based on the hybridization recognition of a single-stranded DNA (ssDNA) probe immobilized onto a gold electrode to its complementary ssDNA is presented. The DNA probe is bound on gold surface electrode by using self-assembled monolayer (SAM) technology. An optimized mixed SAM with a blocking molecule preventing the nonspecific adsorption on the electrode surface has been prepared. In this paper, a DNA biosensor is designed by means of the immobilization of a single stranded DNA probe on an electrochemical transducer surface to recognize specifically Escherichia coli (E. coli) 0157:H7 complementary target DNA sequence via cyclic voltammetry experiments. The 21 mer DNA probe including a C6 alkanethiol group at the 5' phosphate end has been synthesized to form the SAM onto the gold surface through the gold sulfur bond. The goal of this paper has been to design, characterise and optimise an electrochemical DNA sensor. In order to investigate the oligonucleotide probe immobilization and the hybridization detection, experiments with different concentration of DNA and mismatch sequences have been performed. This microdevice has demonstrated the suitability of oligonucleotide Self-assembled monolayers (SAMs) on gold as immobilization method. The DNA probes deposited on gold surface have been functional and able to detect changes in bases sequence in a 21-mer oligonucleotide.     

Rapid electrochemical detection of polyaniline-labeled Escherichia coli O157:H7

There is a high demand for rapid, sensitive, and field-ready detection methods for Escherichia coli O157:H7, a highly infectious and potentially fatal food and water borne pathogen. In this study, E. coli O157:H7 cells are isolated via immunomagnetic separation (IMS) and labeled with biofunctionalized electroactive polyaniline (immuno-PANI). Labeled cell complexes are deposited onto a disposable screen-printed carbon electrode (SPCE) sensor and pulled to the electrode surface by an external magnetic field, to amplify the electrochemical signal generated by the polyaniline. Cyclic voltammetry is used to detect polyaniline and signal magnitude indicates the presence or absence of E. coli O157:H7. As few as 7CFU of E. coli O157:H7 (corresponding to an original concentration of 70 CFU/ml) were successfully detected on the SPCE sensor. The assay requires 70 min from sampling to detection, giving it a major advantage over standard culture methods in applications requiring high-throughput screening of samples and rapid results. The method can be performed with portable, handheld instrumentation and no biological modification of the sensor surface is required. Potential applications include field-based pathogen detection for food and water safety, environmental monitoring, healthcare, and biodefense.     

Monoclonal antibodies against the lac carrier protein from Escherichia coli. 1. Functional studies

The effects of various monoclonal antibodies against purified lac carrier protein on carrier-mediated lactose transport were studied in right-side-out membrane vesicles and in proteoliposomes reconstituted with purified lac carrier protein. Out of more than 60 monoclonal antibodies tested, only one antibody, designated 4B1, inhibits transport. Furthermore, the nature of the inhibition is highly specific in that the antibody inhibits only those transport reactions that involve net proton translocation (i.e., active transport, carrier-mediated influx and efflux under nonenergized conditions, and lactone-induced proton influx). In contrast, the antibody has little effect on equilibrium exchange and no effect on generation of the proton electrochemical gradient or on the ability of the carrier to bind a high-affinity ligand. Clearly, therefore, the antibody alters the relationship between lactose and proton translocation at the level of the lac carrier protein. When entrance counterflow is studied with external [1-14C]lactose at saturating and subsaturating concentrations, it is apparent that antibody 4B1 mimics the effects of deuterium oxide [Viitanen, P., Garcia, M.L., Foster, D.L., Kaczorowski, G. J., & Kaback, H.R. (1983) Biochemistry 22, 2531]. That is, the antibody has no effect on the rate or extent of counterflow when external lactose is saturating but stimulates the efficiency of counterflow when external lactose is below the apparent Km. It seems likely, therefore, that the antibody either inhibits the rate of deprotonation or alters the equilibrium between protonated and deprotonated forms of the carrier. Monovalent Fab fragments prepared from antibody 4B1 inhibit transport in a manner that is similar qualitatively to that of the intact antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amperometric immunosensor for the detection of Escherichia coli O157:H7 in food specimens

A novel, label-free amperometric immunosensor has been developed for the rapid detection of heat-killed Escherichia coli O157:H7 (E. coli O157:H7). This immunosensor was prepared as follows. First, the long-chain, amine-terminated alkanethiol 11-amino-1-undecanethiol hydrochloride (AUT) was self-assembled onto a gold electrode surface to form an ordered, oriented, compact, and stable monolayer possessing -NH(2) functional groups that could immobilize massive gold nanoparticles (GNPs). Next, chitosan-multiwalled carbon nanotubes-SiO(2)/thionine (CHIT-MWNTs-SiO(2)@THI) nanocomposites and GNPs multilayer films were prepared via layer-by-layer (LBL) assembly. The surface area enhancement from the LBL assembly of the multilayer films improves the stability of the immobilized CHIT-MWNTs-SiO(2)@THI. More important, the sensitivity and stability of the immunosensor can be enhanced proportionally to the quantity of the THI mediator immobilized on the electrode surface. Finally, the E. coli O157:H7 antibody (anti-E. coli O157:H7) was covalently bound to the GNP monolayer and its bioactivity was measured by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was employed to characterize the morphology of the MWNTs, CHIT-MWNTs, and CHIT-MWNTs-SiO(2)@THI. Under optimal conditions, the calibration curve for heat-killed E. coli O157:H7 has a working range of 4.12×10(2)-4.12×10(5) colony-forming units (CFU)/ml, and the total assay time was less than 45 min.     

Monoclonal antibodies as probes for detecting lipopolysaccharide expression on Escherichia coli from different growth conditions.

Monoclonal antibody (mAb) probes were used to investigate the expression of lipopolysaccharide (LPS) on four Escherichia coli strains, grown under a variety of conditions in batch culture which mimicked some of the in vivo environmental conditions of an infected host. Techniques of silver staining, immunoblotting, whole cell ELISA and flow cytometry were all used to monitor the expression of LPS on the bacteria and the binding of the anti-LPS mAbs. Growth in heat-inactivated sheep serum and magnesium-depleted conditions demonstrated increased expression of LPS core and subsequent increased binding of anti-core mAbs. Magnesium-depleted conditions also resulted in decreased production of O-polysaccharide material. Iron-depleted bacteria showed only minor changes in LPS expression, although increased binding of anti-core mAbs was observed. Nitrogen-deficient/high-carbon conditions, chosen to promote capsule production, resulted in increased expression of O-polysaccharide and decreased binding of anti-core mAbs.     

Magnetoresistive immunosensor for the detection of Escherichia coli O157:H7 including a microfluidic network

A hand held device has been designed for the immunomagnetic detection and quantification of the pathogen Escherichia coli O157:H7 in food and clinical samples. In this work, a technology to manufacture a Lab on a Chip that integrates a 3D microfluidic network with a microfabricated biosensor has been developed. With this aim, the sensing film optimization, the design of the microfluidic circuitry, the development of the biological protocols involved in the measurements and, finally, the packaging needed to carry out the assays in a safe and straightforward way have been completed. The biosensor is designed to be capable to detect and quantify small magnetic field variations caused by the presence of superparamagnetic beads bound to the antigens previously immobilized on the sensor surface via an antibody-antigen reaction. The giant magnetoresistive multilayer structure implemented as sensing film consists of 20[Cu(5.10nm)/Co(2.47 nm)] with a magnetoresistance of 3.20% at 235Oe and a sensitivity up to 0.06 Omega/Oe between 150Oe and 230Oe. Silicon nitride has been selected as optimum sensor surface coating due to its suitability for antibody immobilization. In order to guide the biological samples towards the sensing area, a microfluidic network made of SU-8 photoresist has been included. Finally, a novel packaging design has been fabricated employing 3D stereolithographic techniques. The microchannels are connected to the outside using standard tubing. Hence, this packaging allows an easy replacement of the used devices.     

Electrophoretic mobilities of Escherichia coli O157:H7 and wild-type Escherichia coli strains.

The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased.