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.     

A NEW RAPID TECHNIQUE FOR SALMONELLA ANTIGEN DETECTION USING A PARTICLE CONCENTRATION FLUORESCENCE IMMUNOASSAY (PCFIA)

A fluorescent assay employing polystyrene beads coated with antibody to a common structural antigen (CSA) of salmonellae has been developed to detect the presence of Salmonella. This sensitive technique is very fast, relatively simple and with further testing may be suitable for screening food products as well as clinical diagnostics. Comparison was made with a commercially available Salmonella antigen capture enzyme immunoassay (EIA). As little as one single colony-forming unit (CFU) could be detected by fluorescence after only 16 h of enrichment. the procedure requires only 1 h to run and uses proportionately smaller volumes of sample and reagents than EIA.     

Enzyme immunoassay for detection of Salmonellae in foods.

An enzyme immunoassay was developed to detect Salmonella in foods. Indirect test protocols were developed for use with microtitration plates or Gilford microcuvettes. Samples from enrichment cultures were mixed with H-specific immunoglobulin G and allowed to react; unbound antibody was removed by three 5-min centrifugation washes; goat anti-rabbit antibody conjugated to alkaline phosphatase was added and allowed to react; and unbound conjugate was removed by centrifugation washing as before. Salmonella-positive samples were indicated by the production of a chromogenic reaction product after the addition of alkaline phosphatase substrate. The color could be read visually or quantified by absorbance. Ninety-eight food samples were examined to compare the enzyme immunoassay with enrichment serology, immunofluorescence, and the Food and Drug Administration pure culture technique. The enzyme immunoassay was sensitive and specific, and it possessed advantages over methods currently in use. Furthermore, when the enzyme immunoassay was used to screen preenrichment media, the results indicated that it might be decidedly more sensitive than the conventional pure culture technique.     

A phosphorylcholine idiotype related to TEPC 15 in mice infected with Ascaris suum.

A serum component, binding antigens having phosphorylcholine (PC) determinants were induced in several strains of mice by infection with Ascaris suum. This component was isolated and demonstrated to be an IgM (K) anti-PC antibody having idiotypic determinants in common with the IgA PC-binding myeloma protein TEPC 15. Rabbit anti-idiotypic antisera prepared with this component had idiotypic specificity for TEPC 15 and cross-idiotypic recognition of MOPC 167 and McPC 603, all IgA PC-binding myeloma proteins. The antisera also recognized determinants not present on TEPC 15. IgM and idiotype levels were quantitated by radial immunodiffusion and PC-specific antibody measured by hemagglutination (HA) with sheep erythrocytes coated with pneumococcal-C-polysaccharide. Mean IgM levels ranged from 2.5 to 8.7 mg/ml, idiotype from 0.54 to 5.3 mg/ml; and HA titers from 1:512 to 1:130,000 in different mouse strains. The high PC-specific antibody response was not duplicated by immunization with dead ascaris larvae or by infection with two other nematode species.     

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.     

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.     

Using a Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella Typhimurium in Chicken Carcass

Chicken is one of the most popular meat products in the world. Salmonella Typhimurium is a common foodbome pathogens associated with the processing of poultry. An optical Surface Plasmon Resonance （SPR） biosensor was sensitive to the presence of Salmonella Typhimurium in chicken carcass. The Spreeta biosensor kits were used to detect Salmonella Typhimurium on chicken carcass successfully. A taste sensor like electronic tongue or biosensors was used to basically ＂taste＂ the object and differentiated one object from the other with different taste sensor signatures. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with dif- ferent antisera or other molecular species. The selectivity of the SPR biosensor was assayed using a series of antibody con- centrations and dilution series of the organism. The SPR biosensor showed promising to detect the existence of Salmonella Typhimurium at 1 x 106 CFU/ml. Initial results show that the SPR biosensor has the potential for its application in pathogenic bacteria monitoring. However, more tests need to be done to confirm the detection limitation.     

Rapid screening method for the detection of antimicrobial substances

Bioluminescence is phenomenon where living organisms produce light and this production is directly dependent on metabolic activity of the organism. Genes encoding enzymes, luciferases, responsible for light production can be cloned into indicator strains, thus allowing sensitive detection of antimicrobial activity. This study utilized bacterial luciferase genes cloned into Staphylococcus aureus, Escherichia coli and Salmonella enterica serovar Typhimurium indicator strains and showed that the detection of antimicrobial activity can be obtained already in 2 h without laborious plate counting and overnight incubation. Indicator strains used in the study harboured luxAB genes responsible of producing light as well as luxCDE genes for synthesis of long-chain fatty aldehyde as substrate for light production. As a consequence, no exogenous aldehyde addition was needed allowing stable light production. Furthermore, the method was used for the detection of antimicrobial activity from lactic acid bacteria after the effect of organic acids was eliminated.     

Evaluation of new culture media for rapid detection and isolation of salmonellae in foods.

Conventional methods for Salmonella detection in foods can require up to 6 and at least 4 days. We have observed that the total analysis time can be reduced to 48 h by using Salmosyst broth as a liquid medium for both preenrichment and selective enrichment and Rambach agar (RA), a new selective plate medium. In samples of artificially contaminated ground beef Salmonella enteritidis was detected at a concentration of 0.4 CFU/g (10 CFU/25 g) by both a conventional method and the new method. Of 519 samples of foods for sale, 38 were Salmonella positive by both methods while 471 were negative. Nine samples which were negative by the conventional method were positive by the Salmosyst-RA method, while one sample positive by the first method was negative by the last. Therefore, the Salmosyst-RA method showed 97.9% sensitivity compared with the 81.2% sensitivity of the conventional method. The new method was also highly specific (98% specificity) in presumptive identification of Salmonella colonies. Furthermore, a 6-h preenrichment in Salmosyst broth has been proved sufficient for the repair of heat-injured Salmonella cells and for subsequent recovery by selective enrichment. In conclusion, the Salmosyst-RA method shows several advantages over both conventional and rapid noncultural methods: (i) only two media are required instead of the five media for conventional methods; (ii) in real time it is comparable to other rapid noncultural methods, which require 30 to 31 h; (iii) it is highly sensitive and specific; and (iv) it allows the isolation of Salmonella strains which can be characterized by appropriate phenotypic and genotypic typing methods for epidemiological investigations.     

Allelic variants of rearranged immunoglobulin heavy and light chain genes in hybridoma PTF-02 and parent myeloma

The hybridoma PTF-02 secretes an antibody against pig transferrin. Rearranged genes for heavy and light immunoglobulin chains have been studied in the genomes of this hybridoma and in the parent myeloma P3-X63.Ag8.653. The hybridoma was shown to contain three rearranged allelic variants of the heavy chain gene's locus. The gene H2, responsible for synthesis of the heavy chain of the antibody to transferrin, was transmitted in the hybridoma cell from a lymphocyte. Two other genes (H1 and H3) were found both in the hybridoma and parent myeloma genomes. The gene H1 was identified in MOPC21 myeloma, which is a precursor of the X63.Ag8 descendent line. Rearranged k genes were also identified both in the hybridoma and parent myeloma. A functional (K2) gene and a fetal (F) gene appeared in the hybridoma genome from an antigen-stimulated normal lymphocyte. The fetal gene was lost in the course of continuous cultivation of the hybridoma PTF-02 cell line. The gene K1 was transmitted from the myeloma used for fusion. In such a way, the pedigree of rearranged heavy and light chain genes in the hybridoma PTF-02 was established. The results obtained in this work may be relevant to many hybridomas whose immortalizing fusion partner is a MOPC21 derivative, and allow one to identify and isolate functional variable genes to create recombinant constructions.     

Homologous monoclonal antibodies specific for alpha 1-3 dextran stimulate polyclonal proliferation of Lyb-5+ B cells

In this paper we describe the polyclonal blast response induced in nonimmune murine splenocytes by two homologous alpha 1-3 dextran-binding myeloma protein, J558 and MOPC104E. This stimulation appears to be independent of MHC or IgCh gene complex control, and proceeds entirely in a T-dependent fashion. The responding B cell population appears to belong to the more mature Lyb-5+ subset. This response was elicited by several independently prepared batches of J558 and MOPC104E, each of which was conclusively shown to be free of endotoxin contamination. Experiments are presented that suggest that this stimulation is being mediated via the Fab portion of these two myeloma proteins, in particular, a shared IdX, rather than through their Fc.     

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.     

APPLICATION OF GENE AMPLIFICATION IN CONJUNCTION WITH A HYBRIDIZATION SENSOR FOR RAPID DETECTION OF SALMONELLA SPP. AND FECAL CONTAMINATION INDICATORS IN ANIMAL FEEDS

The rapid detection of pathogenic microbial species in feed is of paramount importance considering its implications for animal production and food safety. To demonstrate the feasibility of rapidly detecting Salmonella spp. and fecal pollution microbial indicators in feed using gene amplification protocols, commercial and mixed feed samples were inoculated with two levels of a marker strain of S. typhimurium. Liquid extracts of the feed samples were used as templates in gene amplification reactions to amplify sequences associated with fecal contamination indicators. The sequence specificity of the amplification products (amplicons) were confirmed using biotin and fluorescein labeled probes in a navel dual probe based hybridization sensor. Using the combination of gene amplification and the hybridization sensor, the presence of sequences associated with fecal contamination were detected in 15 different feed matrices without employing preenrichment steps. Using this detection methodology, fecal pollution can be confirmed in feed at naturally occurring concentrations. The study demonstrates that it is possible to rapidly detect and confirm the presence of pathogenic bacterial genera in feed matrices by combining robust gene amplification reactions with appropriate post amplification detection systems.     

Increased Sensitivity of Immunofluorescent Assay for Salmonella in Nonfat Dry Milk

The necessity of developing a quick, sensitive, and reliable test for Salmonella in nonfat dry milk (NDM) is evident from the recent tracing of Salmonella outbreaks to this product. Normally, coagulation of casein occurs when assaying NDM under regular cultural conditions, raising the possibility of trapped bacteria. After 20 hr of incubation of NDM in preenrichment lactose broth, enrichment was achieved by using Selenite-Cystine Broth. Smears from the enrichment broth were examined by the fluorescent-antibody technique (FAT) with a commercially available polyvalent O globulin conjugated with fluorescein. Standard cultural methods (SCM) were performed for comparison with FAT. Sensitivity of FAT was definitely improved by the use of trypsin. Casein coagulation of NDM can be avoided by addition of trypsin to samples during initial preenrichment in lactose broth. Samples containing approximately one Salmonella per 10 g were easily detected by FAT with the use of trypsin-treated samples. The method required only 42 hr to complete. Additionally, the use of trypsin enhanced recovery of Salmonella by use of SCM, as evidenced by alteration in the observed coliform to Salmonella ratios.     

Evaluation of different malachite green brands in the performance of rappaport's medium

Summary Three malachite green dyes, Merck's concentrated for microscopy and bacteriology (old), Merck's malachite green oxalate (oxal), and Difco's malachite green, were used for preparation of 3 batches of Rappaport's medium. These media were tested for growth of 40 Salmonella serotypes and for inhibition of competing organisms. All Rappaport's media behaved similarly and supported excellent growth of 38 serotypes. When Salmonella cultures were diluted in fecal-saline suspensions and inoculated in all Rappaport media the competing organisms in these suspensions were efficiently inhibited. However, the inhibition of competing organisms was less efficient when Rappaport's media were inoculated with preenrichment cultures of 20 samples of minced meat in buffered peptone water. In this respect, Difco's malachite green was clearly inferior to the other two dyes. It is concluded that Merck's malachite green oxalate is as efficient as the old dye of the same brand which is no longer produced, and can replace it in the preparation of Rappaport's medium.     

Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella bacteria in meat

We developed a 12-h Salmonella detection method, based on 8 h of preenrichment, followed by automated DNA extraction and a sensitive real-time PCR. The method was optimized to obtain the highest possible yield of cells and DNA. The growth of different Salmonella strains in various preenrichment media and the effects of adding growth-promoting and selective reagents were explored, taking into account their PCR compatibility. The effects of (i) analyzing larger volumes (1 to 5 ml) from preenriched samples and introducing wash steps prior to DNA extraction, (ii) regulating the amount of paramagnetic particles (increasing it from 60 to 90 microl) in the DNA extraction, (iii) eluting the DNA in reduced volumes (25 or 50 microl rather than 100 microl), and (iv) increasing the PCR template volume (from 5 to 20 microl) were investigated. After 8 h of preenrichment, buffered peptone water yielded the highest number of salmonellae. When analyzing minced meat samples, positive effects of increasing the initial sampling volume from 1 to 5 ml and increasing the amount of paramagnetic particles to 90 microl were observed. However, washing the pellet and eluting the DNA in reduced volumes (25 and 50 microl) had no positive effects and resulted in decreased reproducibility. Increasing the amount of PCR template DNA from 5 to 20 mul improved the threshold cycle value by approximately 2. The improved 12-h PCR method was successfully compared to a reference culture method with 100 minced meat and poultry samples, with a relative accuracy of 99%, a relative sensitivity of 98%, and a relative specificity of 100%.     

Detection and serogroup differentiation of Salmonella spp. in food within 30 hours by enrichment-immunoassay with a T6 monoclonal antibody capture enzyme-linked immunosorbent assay.

We previously described an antigen capture enzyme-linked immunosorbent assay which makes use of monoclonal antibody T6, which recognizes an epitope on the outer core polysaccharide of Salmonella lipopolysaccharide molecules that is common to almost all Salmonella serovars. In this paper, we show that this assay can detect between 10(5) and 10(7) Salmonella cells per ml even in the presence of excess Escherichia coli. A total of 153 of 154 (99%) serogroup A to E strains and 51 of 78 (71%) serogroup F to 67 strains were reactive as determined by this assay. This corresponds to a detection rate of approximately 98% of all salmonellae known to affect humans. None of the 65 strains of non-Salmonella bacteria tested positive. Taking advantage of the O-factor polysaccharides also present on the antigen captured by the immobilized T6 antibody, we showed that 136 of 154 Salmonella serogroup A to E strains (88%) were correctly differentiated according to their serogroups by use of enzyme conjugates of a panel of O-factor-specific monoclonal antibodies. We evaluated this assay for the detection and serogroup differentiation of salmonellae directly from enrichment cultures of simulated food, eggs, pork, and infant formula milk. All 26 samples which had been contaminated with Salmonella spp. were detected by T6 (100% sensitivity), with only one false-positive result from 101 samples not contaminated by Salmonella spp. (99% specificity). The detection time was substantially reduced to between 17 and 29 h, depending on the enrichment methods used. Since there were no false-negative results, we concluded that this enrichment-immunoassay method can afford rapid screening for Salmonella spp. in food samples.     

Rapid method for detection of Salmonella in milk by surface plasmon resonance (SPR)

The Plasmonic surface plasmon resonance (SPR) device was used to develop a rapid, simple and specific immunoassay for detection of Salmonella in milk. Rapid detection of Salmonella contamination is a major challenge for the food industry. Salmonella contamination is well known in all foods including pasteurised milk. The SPR assay was developed as a sandwich model using a polyclonal antibody against Salmonella as capture and detection antibody. Milk spiked with Salmonella typhimurium cells, killed by thimerosal (1%, w/w) treatment was used. Using the Plasmonic SPR assay it was possible to detect S. typhimurium down to a concentration of 1.25 x 10(5) cells ml(-1) in both milk and buffer system. The results obtained are comparable with existing, approved rapid Salmonella detection techniques. No negative effects on the sensitivity of the assay are encountered due to the milk matrix. Hence, no sample preparation or clean-up steps are required. The sample volume requirement for the assay is only 10 microl. Using the assay S. typhimurium was detected in milk within 1h, whereas the cultural techniques require 3-4 days for presumptive positive isolates and further time for confirmation. The rapid tests require at least 24h for the results. The Plasmonic SPR device operates on the Kretschmann configuration and is a cuvette-based system with the advantage of having eight channels on one single SPR chip.     

Monoclonal antibody for calcitriol (1 alpha,25-dihydroxyvitamin D3)

Hybridoma cell lines secreting antibodies for vitamin D3 metabolites have been generated by fusing splenocytes from BALB/c mice immunized with 3 beta-glutaryl-25-hydroxyvitamin D3 conjugated to bovine serum albumin (3 beta-glu-25-OH-D3-BSA) and Sp2/O-Ag14 myeloma cells. Purification of monoclonal antibodies from culture media or ascites fluids was accomplished by procedures including affinity chromatography on Protein A-Sepharose 4B. Each monoclonal antibody was analyzed as to its affinity and specificity by equilibrium dialysis and an enzyme immunoassay (EIA) based on a double antibody system. It was demonstrated that clone 1C2-60 produced an antibody highly specific to 1 alpha,25-dihydroxyvitamin D3 (calcitriol), and the clone 2B3-66 antibody was reactive to 25-hydroxyvitamin D3 and similar structural compounds. These two monoclonal antibodies produced by 1C2-60 and 2B3-66 were determined to belong to the IgG2a class, and their affinity constants (Ka) with 3 beta-glu-25-OH-D3 were demonstrated to be 3.6 X 10(9) M-1 and 2.9 X 10(9) M-1, respectively, at 4 degrees C. The characteristics of these monoclonal antibodies were compared with those of conventional antibodies raised in mice and rabbits. Finally, by using monoclonal antibody 1C2-60, a sensitive EIA has been developed that can detect 10 pg of calcitriol.     

Direct immunoassay for detection of salmonellae in foods and feeds

A direct enzyme immunoassay (EIA) with polyclonal antibodies was developed for detecting salmonellae in foods and feeds. Salmonella cells were attached firmly to the wells of polystyrene microtitration plates with a capture-antibody technique. Spicer-Edwards anti-H immunoglobulin G was bound to protein A-beta-D-galactosidase to serve as the signal; 4-methylumbelliferyl-beta-D-galactoside was used as the substrate. The sensitivity threshold was 10(7) cells per ml. Direct EIA, indirect EIA, and pure-culture techniques were compared by using 48 samples of naturally contaminated foods and feeds. The direct EIA was more sensitive than the indirect EIA or pure-culture technique. Food samples were analyzed within 3 working days, and 32 samples were tested simultaneously in a single 96-well microtitration plate. False-positive or false-negative results did not pose a problem. This direct EIA is sensitive, rapid, and amenable to automation.