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PubMed1996

The isolation and detection of Escherichia coli O157 by use of immunomagnetic separation and immunoassay procedures.

The use of immunomagnetic separation (IMS) techniques has been reported to reduce the total test time, and improve the sensitivity, of microbiological tests done on foods. This approach is being adopted in epidemiological investigations into suspected foodborne outbreaks of Escherichia coli O157 infection and has gained acceptance by public health laboratories and the food industry. This study demonstrated the ability of a commercially available IMS procedure, Dynabeads anti-E. coli O157, to enable detection of a few cells of E. coli O157 in 25 g of inoculated minced beef, giving results 1 d earlier than a cultural analysis of similar sensitivity. With correct choice of enrichment broths, IMS may increase isolation rate of E. coli O157 compared to that obtained using conventional cultural methods. It is suggested that this may be due to an increase in relative concentration of E. coli O157 compared with the background microflora present in minced beef, which may reduce reliability of non-IMS detection procedures by masking or mimicking target cells on selective/differential solid media. The use of an immunoassay incorporating an IMS step, EHEC-Tek (Organon-Teknika), enabled detection of a few cells of E. coli O157 in 25 g of minced beef. Comparison of the IMS-ELISA with a standard ELISA procedure (Tecra) indicated the sensitivity of the latter system to be greater, perhaps resulting in the higher isolation rate. The use of a method to reliability isolate and detect extremely low levels of E. coli O157 in a food is necessary to aid reduction in the incidence of this most serious of foodborne pathogens.

Animals↗

PubMed1997

Rapid purification of glial cells using immunomagnetic separation.

By purifying glial cells from brain tissue containing a heterogeneous cell population, a number of interactions that define glial cell diversification and function within the central nervous system have been determined. The current methods for purifying glial cells, however, can be time consuming and costly. In the following study we have adapted the technique of immunomagnetic separation to separately enrich 0-2A progenitor cells and astrocytes from the rat central nervous system (CNS). In this procedure, tissue from the CNS was enzymatically dissociated and incubated in a primary antibody specific to a surface antigen found on the target cell type (e.g. A2B5 or RAN-2). The target cells were then immunologically coupled to magnetic beads, which were precoated with a secondary antibody specific to the primary, and then separated out from the heterogeneous cell population using a magnetic field. We found that the immunomagnetic separation procedure, which was completed within 2 h, produced a near pure population of glial cells (> 99%). This was confirmed by the absence of unbound cells in the bead-bound fraction. The identification and viability of bead-bound cells were established by culturing these cells and subsequently examining their morphology and antigenic expression. This study shows that glial cell types can be separated out of brain tissue to near purity using immunomagnetic separation. This simple procedure is reliable, inexpensive, and achieves levels of purity and viability comparable with currently available techniques of immunopanning and fluorescence-activated cell sorting, within a fraction of the time.

Animals↗

PubMed2005

Combination of immunomagnetic separation with real-time PCR for rapid detection of Salmonella in milk, ground beef, and alfalfa sprouts.

A rapid, specific, and sensitive method for detecting Salmonella spp. in pasteurized milk, ground beef, and alfalfa sprouts was developed. The method combined immunomagnetic separation with a real-time PCR assay based on the double-stranded DNA binding dye SYBR Green I. The primers used produced a product with a melting temperature of 87+/-0.5 degrees C during the PCR assay by amplifying a 284-bp sequence from the invasive gene (invA) of Salmonella. The method was successful in detecting 20 Salmonella strains, but the expected PCR product was not formed by any of 11 other bacterial strains. To test this combined method for the monitoring of Salmonella, Salmonella enterica serotype Newport was inoculated into 52 samples each of pasteurized milk, ground beef, and alfalfa sprouts. Following a 10-h nonselective enrichment step in buffered peptone water, cells were removed by immunomagnetic separation and DNA extracted using the High Pure PCR template preparation kit. The DNA produced was used as a template in the real-time PCR assay. When spiked pasteurized milk, ground beef, and alfalfa sprout samples were analyzed by this protocol, an initial inoculum of 1 CFU/ml, 25 CFU/25 g, and 1.5 CFU/25 g, respectively, was detectable within 13 h. These results indicate that the combination of immunomagnetic separation and real-time PCR assay was a highly specific and sensitive method for the rapid detection of Salmonella.

Animals↗

PubMed2001

The optimization of isolation media used in immunomagnetic separation methods for the detection of Escherichia coli O157 in foods.

AIMS: To compare media used in immunomagnetic separation (IMS) techniques for the isolation of Escherichia coli O157 from food. METHODS AND RESULTS: Foods, both naturally contaminated and spiked, with low numbers (< 1 g(-1)) of stressed E. coli O157 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at 30, 37, 40 and 42 degrees C. Following immunomagnetic separation, beads were plated on a range of selective agars. CONCLUSION: BPW supplemented with vancomycin (8 mg l(-1)) incubated at 42 degrees C, followed by IMS and subsequent plating of immunobeads onto cefixime tellurite sorbitol MacConkey agar plus either Rainbow or CHROMagar agars, proved optimum for the recovery of spiked, stressed E. coli O157 in minced beef, cheese, apple juice and pepperoni. The same protocol was optimum for recovery from naturally-contaminated minced beef and cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum protocol would increase isolation rates of E. coli O157 from foods.

Animals↗

PubMed2004

Immunomagnetic separation of pathogenic organisms from environmental matrices.

One of the most difficult challenges in the analysis of environmental samples is to separate the organism of interest from a sample that is high in background debris. Immunomagnetic separation (IMS) is one technique that has been developed to accomplish this in a rapid and reliable assay. Immunomagnetic separation (or biomagnetic separation) involves a superparamag-netic, monodispersed, polystyrene microsphere that is coated with a specific ligand. When added to a heterogeneous target suspension, the microspheres bind to the desired target. Using a powerful magnet, the microsphere-target complex is then removed from the suspension. Many different targets of interest can be isolated with this technique, including fungal/bacterial cells or spores, protozoan parasites, cellular and subcellular material, proteins, and nucleic acid products. This wide range of application makes IMS one of the most versatile techniques available for the purification of target products from heterogeneous sample matrices.

Animals↗

PubMed2000

Development of immunomagnetic separation technique for isolation of Pseudomonas syringae pv. phaseolicola.

Immunomagnetic separation technique was developed for specific detection and selective isolation of Pseudomonas syringae pv. phaseolicola, the agent of halo-blight disease of beans. Whole-cell and exopolysaccharide fraction of the bacterium was used for polyclonal antibody production in rabbits. High specificity of the antisera was determined in agglutination reactions. The optimum immunocapture time for both antisera was determined as 1 h by using 1/nL CFU (i.e. 10(6) CFU per mL). No significant difference was observed in the binding capacity of cells to immunomagnetic particles with different antisera.

Agglutination Tests↗

PubMed1997

Immunomagnetic separation for enhanced flagellar antigen phase inversion in salmonella.

Immunomagnetic separation (IMS) was used to reduce delays in serotyping caused by slow flagellar phase inversion of diphasic salmonellas. Some 375 strains of 11 salmonella serotypes were examined using IMS. The mean time for successful flagellar phase inversion was reduced from 4.3 (range 1-18) d to 1.0 (range 1-3) d using the IMS method, and in a further experiment phase inversion was achieved within 8 h in 112 of a further 117 strains representing four salmonella serotypes.

Animals↗

PubMed2004

Discontinuous Percoll gradient centrifugation combined with immunomagnetic separation obviates the need for erythrocyte lysis and yields isolated eosinophils with minimal granule abnormalities.

Isolated blood eosinophils are routinely used to study eosinophil activation mechanisms. However, as revealed by ultrastructural analysis, different isolation protocols may yield purified eosinophils with marked variability in granule electron density. In this study, using eosinophil peroxidase (EPO) histochemistry and transmission electron microscopy (TEM), we have compared the morphology of eosinophils in immediately fixed whole blood (to represent a morphological baseline) with isolated eosinophils purified by a number of protocols. Eosinophils in whole blood contained intact specific secondary granules of which a few exhibited marginal coarsening of matrix electron density (4% (95% CI: 2 to 7) altered granules per eosinophil). By contrast, eosinophils purified according to standard protocols, which included erythrocyte lysis with either ammonium chloride or distilled water, showed moderate to extensive loss in density of secondary granule core and/or matrix (NH4Cl: 62% (95% CI: 58 to 66); dH2O: 37% (95% CI: 30 to 44) altered granules). Stepwise analysis of eosinophils during the cell separation processes indicated that the granule abnormalities seen following erythrocyte lysis were further increased following immunomagnetic separation. However, when erythrocyte lysis was omitted, by use of a two-layered Percoll gradient (1.076 g/ml/1.088 g/ml) to which diluted whole blood was applied directly, eosinophils with minimal granule abnormalities (11% (95%CI: 9 to 13) altered granules) could be obtained after immunomagnetic separation. In conclusion, to obtain eosinophils with granule morphology more closely resembling the whole blood baseline phenotype, erythrocyte lysis should be avoided when separating eosinophils from human blood. Thus it will be possible to study in vitro the early transformation of resting eosinophils into the degranulating phenotype found in diseased tissues.

Centrifugation, Density Gradient↗

PubMed1995

Sensitive detection of group A rotaviruses by immunomagnetic separation and reverse transcription-polymerase chain reaction.

An immunomagnetic separation (IMS) method was developed for concentrating rotaviruses from environmental samples, as well as a reverse transcription-polymerase chain reaction (RT-PCR) for the sensitive and specific detection of group A rotaviruses. Magnetic beads were coated with monoclonal antibodies directed against the group-specific, inner capsid protein (VP6) and subsequently used to capture and purify the virus with the help of a magnet. The genome was made available for RT by heat-disrupting the viral particles. A single 40-cycle PCR was as sensitive as a nested PCR, both detecting 0.005 PFU of the Wa strain, corresponding to approximately 5 particles as indicated by EM. The nested PCR was positive for all the group A strains tested, but negative for group C rotaviruses and other RNA viruses. The IMS-RT-PCR method functioned satisfactorily with virus seeded out in fresh water samples; with sea water, the IMS removed most, but not all, inhibiting activity.

Antigens, Viral↗

PubMed2005

Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR.

A method combining immunomagnetic separation (IMS) and real-time (5'-nuclease) PCR was developed to detect Escherichia coli O157:H7. Monoclonal antibody specific for the E. coli O157 antigen was added to protein A-coated magnetic particles to create antibody-coated beads. The beads specifically captured E. coli O157:H7 from bacterial suspensions. The cells were eluted from the beads and lysed by heating; the eluate was then assayed by real-time PCR, using primers and probe specifically targeting the eaeA gene of E. coli O157:H7. Approximately 50% of the cells in suspension were captured by the beads and detected by real-time PCR. No cross-reactivity was detected when other strains of E. coli were tested. This method was applied to detect E. coli O157:H7 from ground beef. Both cell capture efficiency and real-time PCR efficiency were reduced by meat-associated inhibitors. However, we were still able to detect up to 8% of E. coli O157:H7 from inoculated ground beef samples. The detection sensitivity varied among ground beef samples. The minimum detection limit was <5x10(2) cells ml(-1) for suspensions of E. coli O157:H7 in buffer and 1.3x10(4) cells g(-1) for E. coli O157:H7 in ground beef. The combination of IMS and real-time PCR results in rapid, specific and quantitative detection of E. coli O157:H7 without the need for an enrichment culture step.

Adhesins, Bacterial↗

PubMed2006

Comparison of two immunomagnetic separation technologies to deplete T cells from human blood samples.

The objective of this study was to compare the performance of two immunomagnetic separation technologies to deplete T cells from buffy coats of human blood. Specifically, two versions of the commercial MACS(R) Technology: MiniMACS and SuperMACS, and a prototype, flow-through system, the QMS, were evaluated. Peripheral blood mononuclear leukocytes (PBL) were isolated from buffy coats and an immunomagnetic separation of CD3(+) cells was conducted using company and optimized labeling protocols. To mimic peripheral blood containing bone marrow purged hematopoietic stem cells, HSC, CD34 expressing-cells (KG1a) were spiked into PBL prior to T-cell depletion once optimized depletion conditions were determined. Once the labeling protocol was optimized, the MiniMACS system performed well by producing a highly enriched CD3(+) fraction, and a respectable level of depletion of T cells and recovery of KG1a cells in the depleted fraction; an average log(10) depletion of T cells of 2.88 +/- 0.17 and an average recovery of the KG1a cells of 60.8 +/- 5.94% (n = 14). The performance of the SuperMACS system was very similar with an average log(10) depletion of T cells of 2.89 +/- 0.22 and an average recovery of KG1a of 63.1 +/- 8.55% (n = 10). In contrast, the QMS system produced an average log(10) depletion of T cells of 3.98 +/- 0.33 (n = 16) with a corresponding average recovery of 57.9 +/- 16.6% of the spiked CD34+ cells. The aforementioned QMS performance values were obtained using sorting speeds ranging from 2.5 x 10(4) to 1.7 x 10(5) cells per second. It is suggested that the lack of a 100% recovery of the unlabeled KG1a cells is the result of a previously reported "drafting" phenomena which pulls unlabeled cells in the direction of the magnetically labeled cells thereby resulting in loss of the unlabeled cells.

Antigens, CD34↗

PubMedDate not supplied

Performances of the immunomagnetic separation method for Cryptosporidium in water under various operation conditions.

Immunomagnetic separation (IMS) has been specified as a standard method for the measurement of Cryptosporidium in some countries. In this study, the IMS method was evaluated on the basis of the recovery efficiencies of Cryptosporidium oocysts at various IMS operation conditions. The average recovery for different Cryptosporidium concentrations in deionized water was 82.6 +/- 18.2% (n = 52). No significant change in recovery was observed by altering the debris ratio of the water samples. The efficiency was increased by prolonging the reaction time, and by increasing the amount of immunomagnetic beads. The recoveries of oocysts seeded in an Eppendorf with a small reaction volume were similar to those seeded in glass tubes with 10 times the reaction volume. The recovery efficiency of oocysts was reduced significantly when the reaction buffer was replaced by PBS. In conclusion, this method has good reproducibility and high recovery.

Animals↗

PubMed1997

An integrated system using immunomagnetic separation, polymerase chain reaction, and colorimetric detection for diagnosis of Plasmodium falciparum.

An integrated system for sample preparation and DNA detection of the malaria parasite using immunomagnetic separation in combination with the polymerase chain reaction (PCR) and colorimetric analysis is described. A cocktail of three monoclonal antibodies towards merozoite surface antigen-1 was used for magnetic capture of parasites from microliter amounts of whole blood. A sensitivity down to a parasitemia of 10(-6)% was achieved using cultured parasites as a model. The integrated approach was evaluated in a field study. A total of 410 blood samples from patients attending malaria clinics in Trat province and Kanchanaburi province in Thailand were analyzed. The samples were processed by immunomagnetic separation and transferred to central laboratory for PCR-based detection. Microscopic examinations on blood smears were done in parallel; 53% were positive using the DNA-based assay, while only 32% were positive using conventional microscopic analysis. This field study suggests a possible model for large-scale testing of malaria with an increased sensitivity compared with conventional methods.

Animals↗

PubMed1999

Evaluation of immunomagnetic separation and plating media for recovery of Salmonella from meat.

Immunomagnetic separation (IMS) was compared with selective enrichment in selenite cystine (SC) broth for isolation of Salmonella from 86 artificially contaminated ground beef samples. Both Rambach agar (RA) and Hektoen enteric (HE) agar were used as selective plating media. The highest count of Salmonella colonies per plate was obtained after enrichment in SC broth and plating on RA (mean value: 111.1+/-58.1 CFU per plate); the lowest count was obtained after IMS and plating on HE agar (mean value: 65.4+/-36.6 CFU per plate). Salmonella in preenrichment was concentrated 1.7-fold by IMS and represented 31% of the microbial population captured by the beads, but nonspecific binding was high. As a result of the large numbers of competing bacteria, isolations on both RA and HE agar following IMS were quite difficult (mean value for Salmonella colonies: 79.9+/-42.7 CFU per plate; mean value for non-Salmonella colonies: 144.1+/-75.7 CFU per plate; ratio of Salmonella to non-Salmonella colonies: 0.8). This study indicates that SC broth is superior to IMS in the isolation of Salmonella from raw ground meat.

Animals↗

PubMed1999

Rapid detection of Salmonella in chicken washes by immunomagnetic separation and flow cytometry.

Use of flow cytometry to rapidly detect Salmonella in chicken carcass washes was investigated. A direct immunomagnetic separation method was used to prepare samples and was found to be an effective method for separating target cells from debris in chicken carcass washes. When flow cytometry was combined with immunomagnetic separation, the average lowest detectable level of Salmonella detected was 2.3 x 10(4) CFU/ml. Fifty of 100 wash samples from six groups were inoculated with 2 x 10(-1) CFU of Salmonella Typhimurium per milliliter. After 18 h of enrichment at 37 degrees C, all samples were tested for Salmonella using flow cytometry and conventional culture methods. An identification correlation of 96% was found between flow cytometry and xylose-lysine-tergitol agar plating. Quantitative analysis established a significant linear relationship between the enumeration results of flow cytometry and xylose-lysine-tergitol agar plate counts (R2 = 0.796). Time required for flow cytometry, including sample processing and flow cytometric analysis, was less than 1 h.

Animals↗

PubMed2000

[Evaluation of enrichment cultures for detection of Escherichia coli O157 from human stool specimens by using immunomagnetic separation].

We investigated the conditions of enrichment cultures preceding the immunomagnetic separation (IMS) procedure to detect Escherichia coli O157 (E. coli O157) from human stool specimens in routine laboratory examinations. Samples were made by adding either of the three selected strains of E. coli O157 to stools from three healthy human subjects in three different doses. The enrichment cultures were done for 18 hours at 37 degrees C or 42 degrees C, using five different media such as trypticase soy broth (TSB). TSB containing cefixime, tellurite and vancomycin, modified EC broth (mEC), mEC containing novobiocin (N-mEC) and BGLB. The IMS procedure following enrichment culture increased the detection rate of E. coli O157, irrespective of the kinds of the media and the temperatures. It recovered E. coli O157 in 42 samples out of 90, while only 31 samples were positive when the IMS was not applied. The N-mEC showed the best recovery rate of the five enrichment media, and it was the only media that recovered the E. coli O157 Gunmma 298 strains at a level of 2-3 cells per ml. In 73 stool samples collected from probable patients with E. coli O157 infection and subjects who made close contact with the patients, positive results were obtained in six samples with the N-mEC enrichment followed by the IMS procedure, while only three samples were positive by the direct isolation culture. It was concluded, therefore, that, in routine laboratory examinations of E. coli O157 from human stools, the N-mEC enrichment culture for 18 hours followed by the IMS procedure is a sensitive method even when the dose of E. coli O157 in the stool is minimal.

Culture Media↗

PubMed2002

Detection of encephalomyocarditis virus in clinical samples by immunomagnetic separation and one-step RT-PCR.

A method of immunomagnetic separation and one-step reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of Encephalomyocarditis virus (EMCV). EMCV was captured from sample on magnetic beads with homologous monoclonal antibody and then heat denatured. The heated beads were used directly in one-step RT-PCR reaction to amplify a 285-bp PCR fragment at the 3' end of the genomic region that encodes the viral polymerase. This method detected as little as 3.5 TCID(50) of EMCV from infected cell culture. It was shown with this method that the sensitivity of RT-PCR increased when applied for the detection of EMCV added to fecal extract. Using this protocol EMCV was detected from heart homogenate samples containing less than 100 TCID(50)/ml. The amplified product was sequenced to ensure specificity. The immunomagnetic-RT/PCR procedure described here should be useful for the rapid, specific and sensitive detection of EMCV in clinical samples. This technique is rapid, reliable and can be readily adapted to detect EMCV from other clinical samples.

Animals↗

PubMed1998

Immunomagnetic separation of Cryptosporidium parvum from source water samples of various turbidities.

Immunomagnetic separation (IMS) procedures which specifically capture Cryptosporidium oocysts and have the potential to isolate oocysts from debris have become commercially available. We compared two IMS kits (kit DB [Dynabeads anti-Cryptosporidium; product no. 730.01; Dynal A.S., Oslo, Norway] and kit IC1 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC, Portland, Maine]) and a modification of kit IC1 (kit IC2 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC]) at three turbidity levels (50, 500, and 5,000 nephelometric turbidity units [ntu]) by using water matrices obtained from different geographical locations. In deionized water, kit DB yielded recoveries between 68 and 83%, whereas the recoveries obtained with kits IC1 and IC2 were more variable and ranged from 0.2 to 74.5%. In water matrices with turbidity levels up to 500 ntu, the oocyst recoveries were more variable with kit DB; however, the recoveries were similar to those obtained in deionized water. In contrast, there were notable reductions in oocyst recoveries in the turbid matrices with kits IC1 and IC2, and the highest recovery (8.3%) was obtained with a 50-ntu sample. An examination of the effects of age on oocyst recovery with kit DB revealed that oocysts up to 16 weeks old yielded recoveries similar to the recoveries observed with fresh oocysts. These data indicate that all IMS kits do not perform equally well, and it is important to conduct in-house quality assurance work before a commercially available IMS kit is selected to replace flotation procedures for recovery of Cryptosporidium oocysts.

Animals↗