Rapid detection of salmonellae by immunoassays with titanous hydroxide as the solid phase.

Radioimmunometric and enzyme-immunometric assays were developed for the detection of salmonellae in pure and mixed cultures as well as in 59 food samples. The performances of titanous hydroxide suspension and microtiter plates as the solid phase for the immobilization of microorganisms were compared in these immunoassays. Detection of populations of salmonella cells in pure culture, diluted with saline, was 4- to 10-fold more sensitive with the microtiter plates. However, with mixed culture of salmonella and other enterobacterial species, the detection sensitivity with titanous hydroxide was 100- to 160-fold more sensitive than with microtiter plates. Good correlation existed between results of a standard cultural method for the detection of salmonellae in foods and those obtained from radioimmunometric and enzyme-immunometric assays utilizing titanous hydroxide. However, a high incidence of false-positive and false-negative results with food samples occurred with the enzyme-immunometric assay utilizing microtiter plates. The results provided strong evidence for the merits of substituting titanous hydroxide for microtiter plates as the solid phase for the immobilization of salmonellae for their detection by immunoassays. The immunoassays were rapid and enabled the analysis of a large number of selective enrichment cultures of food samples for salmonellae within 8 h.     

Microbially mediated growth suppression and death of salmonella in composted sewage sludge

The role of compost microflora in the suppression of salmonella regrowth in composted sewage sludge was investigated. Microbial inhibition studies of salmonella growth were conducted on nutrient agar, in composts that had been subjected to different temperatures in compost piles, and in radiation sterilized composts inoculated with selected fractions of the compost microflora. Agar assays of inhibition indicated that bacteria and actinomycetes were not suppressive to salmonellae, but a few fungi were. However, compost inoculation assays showed consistently that fungi were not suppressive, but bacteria and actinomycetes were. In compost inoculation assays, microbial antagonists, when present, either killed salmonellae or reduced their growth rate. No suppression of salmonellae occurred in compost taken from 70°C compost-pile zones despite the presence and growth of many types of microbes. With greater numbers and kinds of microbes in 55°C compost, salmonella growth was suppressed 100–10,000-fold. Salmonellae died when inoculated into compost from unheated zones (25–40°C) of piles. Prior colonization of compost with only noncoliform gram-negative bacteria suppressed salmonellae growth 3,000-fold. Coliforms when inoculated prior to salmonellae accounted for 75% of salmonella die-off. Mesophilic curing to allow colonization of curing piles in their entirety by gram-negative bacteria, especially coliforms, should be an effective way to prevent repopulation by salmonellae.     

The Use of Enrichment Serology for Salmonella Detection in Human Foods and Animal Feeds

S ummary . Samples (2208) of food raw materials and products were examined for the presence of salmonellae by use of conventional salmonella detection procedures and the enrichment serology (ES) techniques described by Sperber & Deibel (1969); 348 samples were positive for salmonellae by the conventional procedures. Using the ES technique with a 24 h elective enrichment step, 93–98% of samples positive by the conventional procedures were also positive by the ES technique. Selective enrichment of food samples using tetrathionate broth containing novobiocin, incubated at 41°, led to the best recovery of salmonellae by both the conventional and ES techniques.     

A high-capacity streptavidin-coated microtitration plate

A majority of current immunoassays rely on capturing a specific analyte on a solid phase to allow the separation of the bound analyte from nonbound components. Streptavidin-coated microtitration plates are widely used for immobilization of capturing antibodies, since they provide a generic surface for immobilization of any biotinylated molecule and preserve biomolecule activity much better than direct passive adsorption. Our trials to further improve the properties of the plates resulted in a development of a modified plate, which has higher binding capacity than currently used control plate. The modified coat was prepared by cross-linking streptavidin chemically prior to adsorption onto the microtitration well surfaces. The binding capacities of the plates were measured with biotinylated, europium-labeled molecules and labeled antigen. The immunoassay performance of the plates was studied with noncompetitive, sandwich-type assays of prostate specific antigen (PSA) and human chorionic gonadotropin (hCG). The maximum immobilization capacity of the modified plate was up to 2.5 times higher than that of the control plate. The higher binding capacity was especially emphasized with small-size molecules. The modified high capacity plate increased the linear ranges of the immunoassays and thus delayed the high-dose hook effect. At high antigen concentrations the signal increased up to 59%, and at the conventional linear ranges of the assays, the increase was up to 29%. We conclude that the modified coating method will be valuable for the future miniaturized systems, where high immobilization capacity is needed at limited areas.     

Liposome polymerase chain reaction assay for the sub-attomolar detection of cholera toxin and botulinum neurotoxin type A

We describe an ultrasensitive immunoassay for detecting biotoxins that uses a liposome with encapsulated DNA reporters, and ganglioside receptors embedded in the bilayer, as the detection reagent. After immobilization of the target biotoxin by a capture antibody and co-binding of the detection reagent, the liposomes are ruptured to release the reporters, which are quantified by real-time polymerase chain reaction. The new assays for cholera and botulinum toxins are several orders of magnitude more sensitive than current detection methods. A single 96-well microtiter plate can analyze approximately 20 specimens, including calibration standards and controls, with all measurements conducted in triplicate. Using pre-coated and blocked microtiter plates, and pre-prepared liposome reagents, a liposome polymerase chain reaction assay can be carried out in about 6 h.     

A novel microtiter plate based method for identification of B‐cell epitopes

A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B‐cell epitopes in two different proteins using a peptide‐based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15‐mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor‐α, respectively, was synthesized. The peptides were immobilized covalently using the new, specialized microtiter plates or non‐covalently using conventional ELISA microtiter plates of the high binder type. Subsequently, specific antisera to ubiquitin or murine tumor necrosis factor‐α were added to identify potential linear B‐cell epitopes. All peptides, which were recognized on the conventional microtiter plates, were also recognized on the plates with the covalently bound peptides. In addition, the covalent immobilization method revealed epitopes that were not identified using the method for non‐covalent binding although the peptides were in fact present on the non‐covalent binding surface. The interaction with the hydrophobic surface of the conventional microtiter plate apparently interfered negatively with antibody recognition. The covalently binding microtiter plates described here could be useful for identification of new B‐cell epitopes in protein antigens. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Rapid parallel detection of hygienically relevant microorganisms in water samples by PCR and specific hybridization in microtiter plates

A molecular biological test protocol for the parallel detection of enterococci and Pseudomonas aeruginosa in drinking water was developed. Amplicons labelled with digoxigenin during PCR were hybridized to specific 23S rDNA targeted oligonucleotide probes immobilized in microtiter plates. Detection was performed by addition of anti-digoxigenin-peroxidase-conjugate and chromogenic substrate. Specificity of the probes was evaluated by using pure cultures. First evaluation data with natural water samples in comparison to conventional microbiological analysis according to the German Drinking Water Regulation showed good agreement. Its feasible and rapid performance should be advantageous for use in routine drinking water quality control. Further comparative evaluation studies need to be undertaken to determine the true applicability for routine testing of water samples.     

Immobilization of small molecules and proteins by radio-derivatized polystyrene

When molded polystyrene (PS) products (e.g., microtiter plates) or latex particles are irradiated with high-energy (1-10 Mrads) gamma rays in the presence of nonpolymerizable small molecules such as aromatic amines, some of these molecules incorporate into PS, which leads to the formation of radio-derivatized PS (RDPS). Two classes of RDPS can be identified regarding their ability for immobilization of biologically important molecules: 1) reactive RDPS that are able to form covalent bonds with molecules such as proteins without the help of cross-linkers, and 2) functionalized RDPS that can be used for the immobilization of molecules with activators (e.g., carbodiimides) or cross-linkers. The method can be used for the production of low-noise supports for binding assays. Most of the RDPS can be produced without impairment of the optical quality of PS, making derivatized microtiter plates suitable for colorimetric assays. The principle can be applied for the preparation of affinity sorbents, e.g., for high-performance affinity chromatography and for the immobilization of enzymes using latex PS particles.     

Enhanced detection of staphylococcal genomes in positive blood cultures using a polymeric enzyme complex

This article describes a simple and inexpensive signal amplification method, termed polymeric enzyme detection (PED), which permits rapid and sensitive detection of conserved sequences in the tuf gene that identify Staphylococcus genus, conserved sequences in the femB gene that specifically detect Staphylococcus aureus species, and the methicillin resistance gene mecA directly from positive blood culture bottles. Microbe-specific capture probes were immobilized onto microtiter plates or silicon chips. Target sequences and biotin-labeled, target-specific probes were hybridized to complementary capture probes to create a biotin-labeled, surface-immobilized tripartite complex. In a two-step process, signal was amplified by incubating the surface-immobilized biotin with streptavidin followed by the addition of a 500-kDa dextran polymer conjugated with approximately 80 biotins. Signal was then developed by binding of a streptavidin-horseradish peroxidase conjugate followed by incubation with the substrate tetramethylbenzidine. Use of the PED method improved the lower limit of detection 10- to 100-fold in model DNA hybridization assays with limits of detection as low as 1 fmol/L target DNA. This level of sensitivity permits detection of genomic DNA from methicillin-resistant S. aureus positive blood cultures within 25 to 35 min using either a thin film biosensor chip or a microtiter plate-based assay.     

The Examination of Foods for Enterobacteriaceae using a Test of the Type Generally Adopted for the Detection of Salmonellae

S ummary : In countries that have recently increased their bacteriological inspection of foods, many products have shown considerable improvement in microbiological quality; simultaneously, however, discrepancies between salmonella and coli-aerogenes tests, especially on dehydrated and frozen foods, have tended to become more frequent. These discrepancies have been eliminated by applying the following measures: incorporating glucose in culture media so as to reveal all Enterobacteriaceae; placing reliance on growth rather than gas formation so as to avoid missing anaerogenic organisms, and, especially, examining quantities of foods commensurate with those used in salmonella tests.
For this purpose a procedure is recommended in which 10 g of well homogenized food are enriched in 100 ml of buffered brilliant green-bile-glucose broth, with no attention being paid to gas formation; the enrichment cultures are then streaked on to MacConkey's glucose agar. Single colonies so obtained are tested for fermentative attack on glucose and may be further examined for other characteristics. The same enrichment fluid can be used for the so-called 'nonselective pre-enrichment' of samples of food containing salmonellae impaired by periods spent in conditions of low water activity, low pH, etc.     

3D antibody immobilization on a planar matrix surface

The amount of active capture antibodies immobilized per unit square is crucial to developing effective antibody chips, biosensors, immunoassays and other molecular recognition technologies. In this study, we present a novel yet simple method for oriented antibody immobilization at high density based on the formation of an orderly, organized aggregation of immunoglobulin G (IgG) and staphylococcal protein A (SPA). Following the chelation of His-tag with Ni(2+), antibodies were immobilized on a solid surface in a three-dimensional (3D) manner and exposed the analyte receptor sites well, which resulted in a substantial enhancement of the analytical signal with more than 64-fold increase in detection sensitivity. Pull-down assays confirmed that IgG antibody can only bind to Ni(2+) matrix indirectly via a SPA linkage, where the His-tag is responsible for binding Ni(2+) and homologous domains are responsible for binding IgG Fc. The immobilization approach proposed here may be an attractive strategy for the construction of high performance antibody arrays and biosensors as long as the antibody probe is of mammalian IgG.     

Transglutaminase-mediated protein immobilization to casein nanolayers created on a plastic surface

An enzymatic method for covalent and site-specific immobilization of recombinant proteins on a plastic surface was explored. Using Escherichia coli alkaline phosphatase (AP) with a specific peptide tag (MKHKGS) genetically incorporated at the N-terminus as a model (NK-AP), microbial transglutaminase (MTG)-mediated protein immobilization was demonstrated. To generate a reactive surface for MTG, a 96-well polystyrene microtiter plate was physically coated with casein, a good MTG substrate. Successful immobilization of recombinant AP to the nanolayer of casein on the surface of the microtiter plate was verified by the detection of enzymatic activity. Since little activity was observed when wild-type AP was used, immobilization of NK-AP was likely directed by the specific peptide tag. When polymeric casein prepared by MTG was used as a matrix on the plate, the loading capacity of AP was increased about 2-fold compared to when casein was used as the matrix. Transglutaminase-mediated site-specific posttranslational modification of proteins offers one way of generating a variety of protein-based solid formulations for biotechnological applications.     

Isolation of salmonellas by immunomagnetic separation.

Magnetisable particles, coated with anti-salmonella serum, were used to isolate Salmonella livingstone from pure cultures, mixed cultures and food samples. Beads (10(7] were generally incubated with 10(4) Salm. livingstone cells/ml for 60 min at room temperature. The incubation and washing medium (0.01 mol/l phosphate-buffered saline; PBS) contained 0.1% bovine serum albumin (BSA) and 0.1% Tween 20, respectively. This method gave a recovery for Salm. livingstone of 51.0 +/- 7.8%. However, other micro-organisms such as Aeromonas hydrophila interfered with this test because of non-specific reactions (recovery 50.9 +/- 12.7%). These non-specific reactions could be decreased by using 4% skim milk instead of 0.1% BSA in the incubation medium. The ratio of the recovery of Salm. livingstone relative to the recovery of Aer. hydrophila changed from 0.9 when PBS with 0.1% BSA was used, to 13.4 when PBS with 4% skim milk was used. Immunomagnetic separation of Salmonella spp. from food samples offers good prospects for concentrating salmonella cells from heterogeneous bacterial suspensions, such as enrichment broths.     

Development of nonradioactive microtiter plate assays for nuclease activity

We have developed two microtiter plate assays for the detection of DNA cleavage by nucleases, using 3'-biotinylated oligonucleotide substrates. In the covalently linked oligonucleotide nuclease assay (CLONA), the biotinylated substrates are phosphorylated at the 5' end to facilitate their covalent immobilization on CovaLink NH plates. The cleavage of the covalently immobilized substrate by nucleases results in biotin release. The uncleaved substrate molecules are detected with an enzyme-avidin conjugate. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5' end of the 3'-biotinylated DNA strand. The substrate binds specifically to the wells of streptavidin-coated microtiter plates, in which the nuclease reaction takes place. Uncleaved substrate retains the digoxigenin label, which is detected with an enzyme-labeled anti-digoxigenin antibody. We assessed the efficiency of these two assays by measuring S1 nuclease and DNase I activities, and the inhibitory effect of EDTA and aurintricarboxylic acid on the reaction. Both methods are more convenient than the standard radioactive nuclease assay and are suitable for high-throughput screening of potential nuclease inhibitors, nucleases, and catalytic antibodies. The ALONA assay was found to be more sensitive than the CLONA assay, with a performance similar to that of the standard nuclease assay.     

Comparison of PCR, electrochemical enzyme-linked immunosorbent assays, and the standard culture method for detecting salmonella in meat products

An electrochemical enzyme-linked immunosorbent assay (ELISA) coupled with flow injection analysis (ELISA-FIA) and a PCR-based method using ST11 and ST15 primers for detecting salmonellae in meat were evaluated in comparison with the International Organization for Standardization (ISO) culture method. The methods were applied to experimentally contaminated and naturally contaminated meat samples. The results showed that both ELISA-FIA and PCR allowed detection of salmonella in a product contaminated with a low number of the microorganisms (1 to 10 salmonellae/25 g) after only 5 h of incubation of preenrichment broth, and they were just as effective as the ISO method.     

Protein analysis of mammalian cells in monolayer culture using the bicinchoninic assay

We have applied the bicinchoninic acid (BCA) protein assay to rat brain primary astrocyte monolayer cultures growing in multiwell culture plates. The BCA method provides a more rapid and sensitive procedure with greater stability of color than is obtained using the Lowry method. Also, large numbers of samples can be read rapidly at the available wavelengths on an enzyme-linked immunosorbent assay microtiter plate reader. We found, however, artifactually high readings when using isotonic buffered sucrose to wash the cultures followed by sodium hydroxide to solubilize the cell protein. Such a procedure is commonly used for washing monolayer cell cultures in transport and binding studies. This effect was found to be due to hydrolysis of sucrose to the reducing sugar glucose. Use of Triton X-100 eliminated this problem, but this agent only solubilized about 80% of the protein that could be solubilized with sodium hydroxide. Furthermore, the high viscosity of Triton X-100 makes it more difficult to use. We found that washing the cells with isotonic mannitol solution followed by solubilization with sodium hydroxide gave reliable results. The sensitivity and speed of this method makes it suitable for multiple protein determinations in experiments using large numbers of cell culture samples.     

Comparison of PCR, Electrochemical Enzyme-Linked Immunosorbent Assays, and the Standard Culture Method for Detecting Salmonella in Meat Products

An electrochemical enzyme-linked immunosorbent assay (ELISA) coupled with flow injection analysis (ELISA-FIA) and a PCR-based method using ST11 and ST15 primers for detecting salmonellae in meat were evaluated in comparison with the International Organization for Standardization (ISO) culture method. The methods were applied to experimentally contaminated and naturally contaminated meat samples. The results showed that both ELISA-FIA and PCR allowed detection of salmonella in a product contaminated with a low number of the microorganisms (1 to 10 salmonellae/25 g) after only 5 h of incubation of preenrichment broth, and they were just as effective as the ISO method.     

Enzyme-linked immunosorbent assay for detection of Salmonella lipopolysaccharide in poultry specimens

An enzyme-linked immunosorbent assay (ELISA) for detection of salmonellae was developed and evaluated by using artificially contaminated specimens of poultry feed, feces, litter, or carcass rinsings, and naturally contaminated water samples. Specimens containing salmonellae of serogroups B or C2 inhibited the binding of polyvalent anti-O serum to microtiter plate wells coated with lipopolysaccharide of Salmonella typhimurium (serogroup B) or Salmonella albany (serogroup C2), respectively. Treatment of specimens with Rhozyme 41 (a protease) inhibited nonspecific reactions. The ELISA detected 106 of 111 culture-positive specimens contaminated with salmonellae of serogroups B or C2. Nineteen of 20 specimens containing salmonellae of serogroup C1 and all of 36 culture-negative specimens were ELISA negative. All seven water samples that contained salmonellae of serogroups B or C2, including three that were culture positive only after delayed secondary enrichment, were ELISA positive. Seven of the nine water samples that contained salmonellae of other serogroups, and all 38 culture-negative samples, were ELISA negative. The ELISA was simple to perform, produced results in 48 h, and was more economical than culture methods.     

Capillary Tube Catalase Test

A capillary tube procedure for detection of catalase activities of isolated colonies of pure and mixed cultures on nutrient and blood agar plates is proposed.     

Capillary Tube Catalase Test

A capillary tube procedure for detection of catalase activities of isolated colonies of pure and mixed cultures on nutrient and blood agar plates is proposed.