Use of murine myeloma protein M467 for detecting Salmonella spp. in milk.

This investigation introduces the use of an immunoglobulin A mouse myeloma protein for the detection of Salmonella spp. in milk. The immunoglobulin A protein M467 reacts with flagellin from a wide variety of serotypes. Two assays were developed which used an enzyme-linked immunosorbent assay (ELISA) and M467. Alkaline phosphatase was conjugated to M467 (M467-PH), and the presence of Salmonella dublin was detected by a competitive solid-phase ELISA and a membrane filtration ELISA. The competitive assay competed viable Salmonella spp. found in contaminated milk against polymerized flagellin or whole bacteria fixed to polyvinyl plates for binding by M467-PH. The membrane filtration method utilized a hydrophilic membrane for filtering the bacteria, which were then detected by the reaction with M467-PH and substrate. The sensitivity of the competitive solid-phase ELISA was 10(3) bacteria ml-1, whereas the filter membrane assay required the media containing the bacteria to be cultured in enrichment medium for 4 h before the assay to ensure detection. Either assay could be run within a typical 8-h work day. The filter membrane assay was not suitable for milk due to the high level of natural alkaline phosphatase activity in the liquid food.     

Filtration capture immunoassay for bacteria: optimization and potential for urinalysis.

A novel assay utilizing immuno-labeling, filtration, and electrochemistry for the rapid detection of bacteria has been optimized for the detection of Escherichia coli O157:H7. Bacteria were specifically labeled with alkaline phosphatase conjugated polyclonal antibodies and captured on a polycarbonate track-etched membrane filter (0.2 microm pore size). The filter was then placed directly against a glassy carbon electrode, incubated with enzyme substrate, and the product detected by square wave voltammetry. The high speed and capture efficiency of membrane filtration and inherent sensitivity of electrochemical detection produced a 25-min assay with a detection limit of 5 x 10(3) E. coli O157:H7 per ml using a filtration volume of 100 microl (i.e. 500 cells filtered). The labeling, filtration, and electrochemical steps were optimized, and the assay performance using electrochemical and colorimetric detection methods was compared. The assay was used to detect E. coli O157:H7 that was spiked into filter-sterilized urine at clinically relevant concentrations.     

Development of antibody array for simultaneous detection of foodborne pathogens

Pathogenic bacterial contaminations present serious problems for food industry and public health. Rapid, accurate and affordable assays are needed. In this study, antibody arrays to simultaneously detect two foodborne pathogenic bacteria (Escherichia coli O157:H7 and Salmonella spp.) have been developed using chemiluminescent detecting system. Solid supports using nitrocellulose membrane and poly-l-lysine (PLL) glass slide were compared and optimized for antibody array construction. Many parameters including optimal concentrations of antibodies, blocking reagents, assay time, storage time, sensitivity and cross-reactivity were considered during optimization. This study revealed that the PLL slide was a more suitable support due to highly accurate results and the absence of non-specific background. Phosphate-buffered saline (PBS, pH 7.2) and 3% skim milk in PBS buffer were optimal spotting and blocking reagents, respectively. With the same sensitivity for bacterial detection as in a conventional ELISA (10(5)-10(6)CFU/ml for the E. coli O157:H7 and 10(6)-10(7)CFU/ml for Salmonella detections), this antibody array has advantages of a much shorter assay time of 1h and much lower required amounts of antibodies. Moreover, there was no cross-reactivity in the detection among bacteria tested in this study. Bacteria detection in food sample was feasible as demonstrated using bacteria-added milk.     

M467: a murine IgA myeloma protein that binds a bacterial protein. I. Recognition of common antigenic determinants on Salmonella flagellins.

We have studied the binding of M467, an IgA murine myeloma protein, to flagellin from seven species of Salmonella. It was found that M467 was reacting with antigenic determinants that were common to all the flagellins studied. These determinants were not related to serotypic antigens. Electronmicrographs of unreduced M467 showed a variety of polymeric species bound to flagella in a manner that could produce immobilization as well as agglutination and precipitation through cross-linking of antigenic determinants. Immunodiffusion in agar gel revealed that M467 was recognizing more than one group of peptide determinants on the flagellins studied. Passive hemagglutination inhibition and a solid phase radioimmunoassay provided evidence that there were differences in binding avidities between M467 and the various Salmonella flagellins studied. It was concluded that M467 is binding more than one specific group of antigenic peptide determinants on flagellin molecules. Flagellin from four of the seven species of Salmonella studied were deficient in one or more of these determinants.     

Detection of Salmonella in food samples by the combination of immunomagnetic separation and PCR assay.

A combination of immunomagnetic separation and polymerase chain reaction (IMS-PCR) was used to detect Salmonella in food samples. Pre-enrichment of samples was combined with filtration through a membrane for the removal of food debris. The IMS-PCR assay combines selective extraction of bacteria by specific antibodies with primer specific PCR amplification that enables to detect Salmonella in non-fatty food samples in 24 h. In comparison with conventional cultural methods, the IMS-PCR is a rapid and specific method. Combined with filtration bags, it partially reduces the negative effects of the food matrix and allows the quick detection of Salmonella cells. The shortened protocols for Salmonella spp. detection described here can improve considerably current methodologies.     

Stimulation of human blood lymphocyte by different polyclonal B cell activators of bacterial and plant origin: Production of IgM,IgG and IgA estimated by the ELISA method

Lymphocytes isolated from peripheral blood of healthy donors were stimulated in vitro with pokeweed mitogen, concanavalin A, flagellin, Nocardia delipidated cell mitogen (NDCM) and heat-killed bacteria Escherichia coli and Actinomyces viscosus. A simple and sensitive technique, enzyme-linked immunosorbent assay (ELISA) was used for the detection of nanogram levels of IgM, IgA and IgC in media from lymphocyte cultures after polyclonal stimulation, Pokeweed mitogen, NDCM and E. coli were shown to stimulate a high production of IgM; after stimulation with A. viscosus a higher production of IgA was detected. No immunoglobulin production was observed after stimulation with polymerized flagellin.     

Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project

A real-time PCR assay using non-patented primers and a TaqMan probe for the detection and quantification of Salmonella spp. is presented. The assay is based on an internationally validated conventional PCR system, which was suggested as a standard method for the detection of Salmonella spp. in the FOOD-PCR project. The assay was sensitive and specific. Consistent detection of 9.5 genome equivalents per PCR reaction was achieved, whereas samples containing an average of 0.95 genome equivalents per reaction were inconsistently positive. The assay performed equally well as a commercially available real-time PCR assay and allowed sensitive detection of Salmonella spp. in artificially contaminated food. After enrichment for 16 h in buffered peptone water (BPW) or universal pre-enrichment broth (UPB) 2.5 CFU/25 g salmon and minced meat, and 5 CFU/25 g chicken meat and 25 ml raw milk were detected. Enrichment in BPW yielded higher numbers of CFU/ml than UPB for all matrices tested. However, the productivity of UPB was sufficient, as all samples were positive with both real-time PCR methods, including those containing less than 300 CFU/ml enrichment broth (enrichment of 5 CFU/25 ml raw milk in UPB).     

Genetically engineered Bifidobacterium animalis expressing the Salmonella flagellin gene for the mucosal immunization in a mouse model

BACKGROUND: A critical component of the host defense against enteric infections is the immunological response of the mucosal membrane, a major starting point of infectious disease, such as typhoid fever. The mucosal immune system consists of an integrated network of lymphoid tissues, mucous membrane-associated cells, and effector molecules. In the present study, we developed a recombinant Bifidobacterium animalis (B. animalis) genetically modified with the Salmonella flagellin gene for mucosal immunization as an oral typhoid vaccine. METHODS: We constructed an oral vaccine against Salmonella typhimurium, consisting of recombinant B. animalis containing the flagellin gene of Salmonella. The recombinant B. animalis was administered orally to mice every other day for 6 weeks. Anti-flagellin antibodies in the serum and stools were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: We detected significantly higher levels of flagellin-specific IgA in the serum and stools of the mice treated with the recombinant B. animalis containing the flagellin gene than was seen in those treated with parental B. animalis. CONCLUSIONS: Our findings suggest that an oral vaccination using recombinant B. animalis genetically modified with the flagellin gene of Salmonella may be effective against Salmonella infections.     

Quantification and specific detection of collagenous proteins using an enzyme-linked immunosorbent assay and an immunoblotting for cyanogen bromide peptides

A method for the detection of collagenous proteins within cyanogen bromide digests of tissues has been devised. The peptides produced by digestion with cyanogen bromide were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter. They were stained on the filter by incubation first with antibodies to collagen and then with a second antibody covalently linked to horseradish peroxidase, 4-chloro-1-naphthol was added, and the bound enzyme was assayed. This procedure is useful for the identification and characterization of collagens of types I, III, IV, and V in tissues. In addition, we have developed a sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) which is convenient for quantifying collagens (types I, III, and IV) in tissues. In this kind of assay, soluble cyanogen bromide peptides compete with cyanogen bromide peptides adsorbed onto a solid-phase support for rabbit anti-collagen antibodies. We determined the amount of bound antibody by using goat anti-rabbit immunoglobulin G covalently conjugated to horseradish peroxidase and then provided a substrate for the enzymatic reaction. The sensitivity range of the ELISA is 0.09 micrograms/ml in the region of 90 to 10% binding.     

Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone

A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples.     

Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone.

A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples.     

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk (r = 0.94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0.1 M, pH 3.0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better (r = 0.90) than that using standard 2 ml samples (r = 0.88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.     

A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens

A major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays.     

A method of solid phase immunoenzyme analysis using antigen molecules immobilized on membranes

The enzyme-linked immunoassay modification has been worked out. The method combines advantages of membrane technology of antigen immobilization which is used in the enzyme immunosensory technique and of conventional enzyme-linked immunosorbent assay. The nitrocellulose and polypropylene membranes are used as a solid-phase. The purified rabbit immunoglobulin G is immobilized on the surface of membranes as the first layer. The competitive immunoassay is employed. The immunoglobulin G concentration range is 1-1000 ng/ml. The membranes with the immobilized antigen can be repeatedly used after incubation in 0.1 M glycine buffer, pH 2.5. The dry membrane with the immobilized antigen can be used after keeping for 6 months in refrigerator at 4 degrees C without changing the concentration range measured.     

Further studies on the feasibility of one-day Salmonella detection by enzyme-linked immunosorbent assay.

A model system previously developed for the rapid detection of Salmonella typhimurium in foods was improved and extended to many other Salmonella serotypes. The original protocol, which consisted of an overnight nonselective culture followed by a specific enzyme-linked immunosorbent assay (ELISA), was modified and improved. A sandwich ELISA which used polyclonal antibodies for the capture stage and a cocktail of monoclonal antibodies for the detector stage was developed. The assay recognized a wide range of Salmonella serotypes; S. enteritidis, the most important serotype in the United Kingdom had a detection limit in the ELISA of about 4 x 10(2) cells ml-1. The cultural stage prior to the ELISA was either a single nonselective broth (incubated for 28 h) or a preenrichment broth (incubated for 7 h) plus a selective broth (incubated for 21 h). Antibodies which bind to cells grown in the unfavorable conditions of a selective medium were selected. It was concluded that, in the future, the shortened protocols for the detection of Salmonella spp. in foods described here will be of considerable value.     

Partial Analysis of the Flagellar Antigenic Determinant Recognized by a Monoclonal Antibody to Clostridium tyrobutyricum

In order to count Clostridium tyrobutyricum spores in milk after membrane filtration, murine 21E7-B12 monoclonal antibody was produced. Elution of the monoclonal antibody from this antigen, the flagellar filament protein, by carbohydrate ligands was used to study the epitope structure. A competitive elution of an anti-dextran monoclonal antibody by carbohydrate ligands served as a control in order to validate the immunological tool applied to flagellin epitope study. The carbohydrate moiety of flagellin contained D -glucose and N-acetyl-glucosamine in a molar ratio of 11:1 as determined by gas-liquid chromatography and 2 low-abundancy unidentified compounds. In ELISA, D -glucose and N-acetyl-glucosamine did not dissociate the antibody-flagellin complex contrary to maltose, maltotriose, maltotetraose and maltopentaose. The efficiency of elution increased from the dimer to the pentamer and became nil for maltohexaose and maltoheptaose. The fact that the hexamer and heptamer could not react with the 21E7-B12 monoclonal antibody could be explained by a drastic conformational change. The overall stretched maltopentaose switch to a helical-shaped maltoheptaose which could not fit the 21E7-B12 monoclonal antibody antigen-combining site. Thus, flagellin epitope may contain α(1→4) linked glucose residues plus either N-actyl-glucosamine or an unidentified compound that maintain it in an extended shape.     

Enzyme immunoassay in which a myeloma protein is used for detection of salmonellae

An enzyme immunoassay (EIA) in which an immunoglobulin A monoclonal antibody from a myeloma (MOPC 467) is used was developed to detect the presence of Salmonella organisms. This myeloma protein binds to a flagellar determinant of the organisms but is not directed toward the H antigens. Of 100 strains tested, 94% were detectable with this antibody. The EIA, used with MOPC 467, is quick, sensitive, and specific, showing virtually no cross-reactivity to other enteric organisms. Initial screening of antibody reactivity was performed by Ouchterlony gel diffusion with the supernatants of heat-treated Salmonella cultures. After this, an EIA was performed on the heat extracts with the myeloma protein, which had been directly coupled to alkaline phosphatase. A positive reaction was indicated by the production of a yellow color after the addition of a substrate (p-nitrophenylphosphate), and this was quantitated by determining the absorbance at 405 nm. The EIA proved to be slightly more sensitive than the Ouchterlony analysis. The sensitivity of the EIA is such that as few as 10(6) Salmonella organisms per ml were detected. This concentration was easily obtained after a 24-h preenrichment incubation of the sample. Mixtures of Salmonella strains with a 10 x concentration of Escherichia coli did not prevent detection of the Salmonella strains. This EIA can be successfully used to detect contamination of foods, as it was used to detect the intentional contamination of infant formula in these studies. Indications are that the EIA is sensitive enough to detect Salmonella strains in M broth subcultures taken directly from a preenrichment culture. Testing of samples could thus be completed 36 h after culture initiation, rather than after 96 h, the time currently needed.     

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk ( r = 0·94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0·1 M, pH 3·0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better ( r = 0·90) than that using standard 2 ml samples ( r = 0·88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.     

Enzyme immunoassay in which a myeloma protein is used for detection of salmonellae.

An enzyme immunoassay (EIA) in which an immunoglobulin A monoclonal antibody from a myeloma (MOPC 467) is used was developed to detect the presence of Salmonella organisms. This myeloma protein binds to a flagellar determinant of the organisms but is not directed toward the H antigens. Of 100 strains tested, 94% were detectable with this antibody. The EIA, used with MOPC 467, is quick, sensitive, and specific, showing virtually no cross-reactivity to other enteric organisms. Initial screening of antibody reactivity was performed by Ouchterlony gel diffusion with the supernatants of heat-treated Salmonella cultures. After this, an EIA was performed on the heat extracts with the myeloma protein, which had been directly coupled to alkaline phosphatase. A positive reaction was indicated by the production of a yellow color after the addition of a substrate (p-nitrophenylphosphate), and this was quantitated by determining the absorbance at 405 nm. The EIA proved to be slightly more sensitive than the Ouchterlony analysis. The sensitivity of the EIA is such that as few as 10(6) Salmonella organisms per ml were detected. This concentration was easily obtained after a 24-h preenrichment incubation of the sample. Mixtures of Salmonella strains with a 10 x concentration of Escherichia coli did not prevent detection of the Salmonella strains. This EIA can be successfully used to detect contamination of foods, as it was used to detect the intentional contamination of infant formula in these studies. Indications are that the EIA is sensitive enough to detect Salmonella strains in M broth subcultures taken directly from a preenrichment culture. Testing of samples could thus be completed 36 h after culture initiation, rather than after 96 h, the time currently needed.     

Monoclonal antibodies that detect live salmonellae.

Nine immunoglobulin G and nine immunoglobulin M murine monoclonal antibody-producing hybridomas reactive with live Salmonella bacteria were obtained from several fusions of immune spleen cells and Sp2/0 myeloma cells. The antibodies were selected by the magnetic immunoluminescence assay. The monoclonal antibodies were reactive with serogroups A, B, C1, C2, D, E, and K and Salmonella choleraesuis subsp. diarizonae. Each monoclonal antibody proved to be reactive with a distinct serotype. Clinical isolates belonging to these Salmonella serogroups could be detected. Reactivity with non-Salmonella bacteria proved to be minor.