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At least 19 recordsLinked to original sources
PubMed1993

Comparison of immunomagnetic beads coated with protein A, protein G, or goat anti-mouse immunoglobulins. Applications in enzyme immunoassays and immunomagnetic separations.

Immunomagnetic beads were prepared using either protein A (PA) or protein G (PG) coupled to magnetic beads for binding antibodies at their Fc region. The performance of these beads was compared with commercially available beads coated with goat anti-mouse (G alpha M) immunoglobulins. Both the PA- and PG-beads possessed a higher binding capacity than the G alpha M-beads for the monoclonal antibodies tested, although, PA bound weakly with some IgG1 antibodies. PA-beads were compared with G alpha M-beads in a magnetic enzyme immunoassay for the detection of mouse immunoglobulins as an alternative to a conventional capture ELISA. The magnetic enzyme immunoassay was characterized by a detection time of less than 60 min and a linear assay range from 5-10 to 500 ng/ml for G alpha M-beads and 5-10 to 1000 ng/ml for PA-beads. The capture ELISA was linear from 10 to 250 ng/ml. For immunomagnetic separation of Salmonella with immunomagnetic beads, PA-beads were superior to both PG- and G alpha M-beads. For specific isolation of bacteria from heterogeneous suspensions by immunomagnetic separation, PA- and PG-beads are preferable since G alpha M-beads crossreact with bacteria possessing proteins with Fc-binding activity.

Animals↗
PubMed1990

Detection of Listeria monocytogenes in foods by immunomagnetic separation.

Immunomagnetic separation with immunomagnetic beads was used to isolate strains of Listeria monocytogenes both from pure cultures and from heterogeneous suspensions. The monoclonal antibodies used recognized all six strains of serotype 4 but only one of three strains of serotype 1. Coating procedure, incubation time, and number of immunomagnetic beads influenced the sensitivity of the isolation method. Less than 1 x 10(2) bacteria per ml in pure cultures and less than 2 x 10(2) bacteria per ml in enriched foods could be detected. The method represents a new approach to extraction and isolation of pathogenic bacteria directly from foods, after resuscitation, or from enrichment broths.

Agglutination Tests↗
PubMed2001

Recovery of Cryptosporidium oocysts and Giardia cysts from source water concentrates using immunomagnetic separation.

Immunomagnetic separation (IMS) procedures for the simultaneous isolation of Cryptosporidium oocysts and Giardia cysts have recently become available. We validated Dynal's GC-Combo IMS kit using source water at three turbidity levels (5000, 500 and 50 nephelometric turbidity units [ntu]) obtained from different geographical locations and spiked with approximately 9--11 (oo)cysts per ml. Mean recoveries of Cryptosporidium oocysts and Giardia cysts in deionized water were 62% and 69%, respectively. In turbid water matrices, mean recoveries of Cryptosporidium oocysts were between 55.9% and 83.1% while mean recoveries of cysts were between 61.1% and 89.6%. Marginally higher recoveries of the heat inactivated (oo)cysts were observed (119.4% Cryptosporidium oocysts and 90.9% Giardia cysts) in deionized water when compared with recoveries of viable (oo)cysts (69.7% Cryptosporidium oocysts and 79% Giardia cysts). Age of (oo)cysts on recoveries using the GC-Combo IMS kit demonstrated no effects up to 20 months old. Recovery of Giardia cysts was consistent for isolates aged up to 8 months (81.4%), however, a significant reduction in recoveries was noted at 20 months age. Recoveries of low levels (5 and 10 (oo)cysts) of Cryptosporidium oocysts and Giardia cysts in deionized water using IMS ranged from 51.3% to 78% and from 47.6% to 90.0%, respectively. Results of this study indicate that Dynal's GC-Combo IMS kit is an efficient technique to separate Cryptosporidium/Giardia from turbid matrices and yields consistent, reproducible recoveries. The use of fresh (recently voided and purified) (oo)cysts, aged (oo)cysts, viable and heat-inactivated (oo)cysts indicated that these parameters do not influence IMS performance.

Age Factors↗
PubMed1998

Isolation of Mycobacterium paratuberculosis from milk by immunomagnetic separation.

An immunomagnetic separation (IMS) technique was developed to facilitate selective isolation of Mycobacterium paratuberculosis cells from milk. Rabbit polyclonal antibodies against radiation-killed intact M. paratuberculosis cells were produced and used to coat sheep anti-rabbit immunoglobulin G (IgG) type M-280 Dynabeads. The rabbit anti-M. paratuberculosis IgG-coated beads (IMB) reacted strongly with laboratory strains of M. paratuberculosis as determined by slide agglutination, and microscopic examination confirmed that M. paratuberculosis cells attached to the IMB. The IMB were found to have a maximum binding capacity of 10(4) to 10(5) CFU of M. paratuberculosis. Studies showed that IMS selectively recovered M. paratuberculosis from inoculated milk containing as few as 10 CFU of M. paratuberculosis per ml when 10 microliter IMB (ca. 10(6) beads) was added to 1 ml of milk and the preparation was incubated for 30 min at room temperature with gentle agitation. Larger volumes of milk (10 and 50 ml) were centrifuged and resuspended in 1 ml of phosphate-buffered saline-0.05% Tween 20 prior to IMS in order to increase the sensitivity of the method. Currently, primary isolation of M. paratuberculosis from a milk sample relies on chemical decontamination, followed by culturing on Herrold's egg yolk medium, which must be incubated at 37 degreesC for up to 18 weeks. The potential value of our IMS method is as an aid for rapid detection of M. paratuberculosis in milk when it is used in conjunction with end point detection methods, such as IS900 PCR or an enzyme-linked immunosorbent assay.

Animals↗
PubMed2006

Improvement of immunomagnetic separation for Escherichia coli O157:H7 detection by the PickPen magnetic particle separation device.

Conventional immunomagnetic separation (IMS) procedures, which use an external magnetic source to capture magnetic particles against the side of a test tube, are labor-intensive and can have poor sensitivity for the target organism because of high background microflora that is not effectively washed away during the IMS process. This report compares the conventional IMS procedure to a new IMS procedure with an intrasolution magnetic particle transfer device, the PickPen. The IMS target for the majority of these studies is Escherichia coli O157:H7 in various types of samples, including cattle feces, hides, carcasses, and ground beef. Comparison of the two IMS methods showed a significant difference (P < 0.05) in the efficiency of detecting E. coli O157:H7 from cattle carcass surface, cattle hide, and cattle fecal samples. No significant improvement (P > 0.05) in E. coli O157:H7 detection was observed when the PickPen IMS procedure was used to isolate this pathogen from ground beef samples. Use of the PickPen IMS greatly increases the throughput of the IMS procedure and may be more compatible with various emerging technologies for pathogen detection. In addition, the efficacy of sequential IMS for multiple pathogens is reported herein.

Animals↗
PubMed1998

Improvement of the immunomagnetic separation method selective for Escherichia coli O157 strains.

Immunomagnetic separation is a useful enrichment method selective for Escherichia coli O157 cells against non-O157 E. coli cells from a preenrichment culture. However, E. coli cells are adsorbed onto a solid surface nonspecifically. With the conventional immunomagnetic separation method, this nonspecific adsorption interfered with immunomagnetic separation. It was found that this interference could be reduced with a low-ionic-strength solution. When immunomagnetic separation was carried out with this solution, the proportion of E. coli O157 cells to non-O157 E. coli cells increased from 9.6 to 31.4 times compared to the proportion obtained by the conventional immunomagnetic separation method. The effectiveness of this solution was successfully evaluated by the use of E. coli O157-spiked samples.

Adsorption↗
PubMed2004

The detection of non-O157 E. coli in food by immunomagnetic separation.

AIMS: To compare immunomagnetic separation (IMS) protocols (enrichment media and temperature) for the isolation of Escherichia coli serotypes O26 and O111 from four different foods. METHODS AND RESULTS: Foods (minced beef, cheese, apple juice and pepperoni) spiked with low numbers (<100 g(-1)) of stressed nalidixic mutant E. coli serotypes O26 and O111 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at temperatures of 37 and 42 degrees C to optimize the IMS technique. BPW enrichments gave increased recoveries of both serotypes compared with tryptone soya and EC broths. Elevated temperatures of incubation at 42 degrees C were superior to 37 degrees C. CONCLUSIONS: Positive detection of low numbers of stressed target pathogens in all replicate tests was only possible using BPW enrichments. The majority of tests from alternative enrichments resulted in zero or single colonies recovered post-IMS. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum IMS protocol would improve isolation rates of E. coli O26 and O111 from foods and lead to increased safety for the consumer. Sub-optimal IMS protocols could lead to foods being incorrectly labelled free from these pathogens.

Beverages↗
PubMed1998

Immunomagnetic separation with mediated flow injection analysis amperometric detection of viable Escherichia coli O157.

The coupling of an immunological separation (using immunomagnetic beads) with amperometric flow injection analysis detection of viable bacteria is presented. Using a solution containing Escherichia coli O157, the electrochemical response with two different mediators [potassium hexacyanoferrate(III) and 2,6-dichlorophenolindophenol] was evaluated in the FIA system. Antibody-derivatized Dynabeads were used to selectively separate E. coli O157 from a matrix. The kinetics and the capacity parameters regarding the attachment of bacteria to the immunobeads were studied. The immunomagnetic separation was then used in conjunction with electrochemical detection to measure the concentration of viable bacteria. A calibration curve of colony-forming units (cfu) against electrochemical response was obtained. The detection limit for this rapid microbiological method was 10(5) cfu mL-1, and the complete assay was performed in 2 h. Some advantages over ELISA methods are the direct detection of viable cells (and not total bacterial load) and the need for only one antibody (not enzyme-labeled), thus making the assay faster (only one washing step is necessary) and less expensive.

2,6-Dichloroindophenol↗
PubMed1996

Immunomagnetic separation can enrich fixed solid tumors for epithelial cells.

Immunomagnetic separation is a highly specific technique for the enrichment or isolation of cells from a variety of fresh tissues and microorganisms or molecules from suspensions. Because new techniques for molecular analysis of solid tumors are now applicable to fixed tissue but sometimes require or benefit from enrichment for tumor cells, we tested the efficacy of immunomagnetic separation for enriching fixed solid tumors for malignant epithelial cells. We applied it to two different tumors and fixation methods to separate neoplastic from non-neoplastic cells in primary colorectal cancers and metastatic breast cancers, and were able to enrich to a high degree of purity. Immunomagnetic separation was effective in unembedded fixed tissue as well as fixed paraffin-embedded tissue. The magnetically separated cells were amenable to fluorescence in situ hybridization and polymerase chain reaction amplification of their DNA with minimal additional manipulation. The high degree of enrichment achieved before amplification contributed to interpretation of loss of heterozygosity in metastatic breast cancers, and simplified fluorescence in situ hybridization analysis because only neoplastic cells were hybridized and counted. Immunomagnetic separation is effective for the enrichment of fixed solid tumors, can be performed with widely available commercial antibodies, and requires little specialized instrumentation. It can contribute to interpretation of results in situations where enrichment by other methods is difficult or not possible.

Breast Neoplasms↗
PubMed1995

Immunomagnetic separation of Salmonella from foods and their detection using immunomagnetic particle (IMP)-ELISA.

An immunomagnetic particle based ELISA (IMP-ELISA) for the detection of Salmonella from foods has been developed using Dynabeads anti-Salmonella (Dynal, Oslo, Norway). Appropriate sample preparation protocols to allow rapid detection of Salmonella serovariants in processed (powdered egg products) and non-processed (raw chicken) samples have been established. Pre-enriched broths of heat processed samples likely to harbour only low levels of competitive enteric flora, were boiled and used directly for IMP-ELISA. For non-heat processed or raw samples likely to contain higher numbers of such competing organisms, live Salmonella cells were first isolated by immunomagnetic separation (IMS) from standard pre-enrichment broths, and then post-selectively enriched for a short time in M-broth followed by boiling before IMP-ELISA. The total assay time including sample preparation was under 26 h for both types of procedure, with a lower detection limit of 10(5) Salmonella cells/ml of sample. In an evaluation of naturally contaminated poultry samples, all 45 of 48 samples previously shown to contain salmonellae in a comparison of ISO, IMS-Plating, Salmonella-Tek ELISA (Organon Teknika, Inc. Durham, NC) and a modification of the latter based on IMS, were identified as positive. None of the other methods gave positives for all 45.

Animals↗
PubMed2005

Multiple sample flow through immunomagnetic separator for concentrating pathogenic bacteria.

The standard method of immunomagnetic separation for isolating pathogenic bacteria from food and environmental matrices processes 1 ml volumes. Pathogens present at low levels (<0.5 pathogenic bacteria/g) will not be consistently detected by this method. Here a multiple sample flow through immunomagnetic separator has been designed and tested to process large volume samples (50 to 250 ml). Preliminary results show >97% recovery of polydisperse magnetic particles (diameter range 1 to 8 microm) containing 29-33% w/w Fe3O4 content. Between 70 and 130 times more of the pathogenic bacteria Escherichia coli O157 is recovered from PBS compared with the standard 1 ml method. Also, the recovery of E. coli O157 from beef mince homogenates, after a 4 h incubation at 42 degrees C, is between 80 and 180 times higher than the standard 1 ml method.

Bacteria↗
PubMed2003

Sensitive and specific detection of circulating cancer cells in patients with hepatocellular carcinoma; detection of human telomerase reverse transcriptase messenger RNA after immunomagnetic separation.

PURPOSE: We evaluated whether detection of human telomerase reverse transcriptase (hTERT) mRNA after immunomagnetic separation is useful to detect circulating cancer (CC) cells. EXPERIMENTAL DESIGN: Two ml of peripheral blood were collected from 55 cases with hepatocellular carcinoma (HCC), 20 cases with chronic liver diseases devoid of cancer, and 20 healthy volunteers. Then 1500 and 500 micro l were subjected to immunomagnetic separations using Ber-EP4 and anti-CD45 antibodies, harvested and supernatant cells were collected as epithelial and nonleukocyte fractions, respectively. Samples of each fraction were subjected to reverse transcription-PCR detecting beta-actin, interleukin-2 receptor (IL-2r), alpha-fetoprotein, and hTERT mRNAs. The cases were judged to be positive, equivocal, or negative for CC cells when hTERT positivity with IL-2r negativity, hTERT positivity with IL-2r positivity, or hTERT negativity was seen in epithelial and/or nonleukocyte fractions, respectively. RESULTS: The dilution experiments revealed that our system could detect 10(0-1) HeLa cells involved in 2 ml of blood. The Ber-EP4-harvested cells from cases with distant metastasis were positive for immunostaining using Hep Par 1 monoclonal antibody. CC cells were judged to be positive in 29 of 55 (53%) HCC cases. On the contrary, no cases without HCC were determined to be positive. The frequency of positivity was significantly correlated with disease extent of HCC. CONCLUSIONS: These results strongly suggest that detection of hTERT mRNA after immunomagnetic separation is a specific and sensitive tool to detect CC cells and that it would provide useful source for further investigation of cancer metastasis.

Aged↗
PubMed1995

An evaluation of immunomagnetic separation for the detection of salmonellas in raw chicken carcasses.

Immunomagnetic separation techniques were used in the isolation of salmonella from raw chicken carcasses. Improved isolation rates were achieved with increased specificity and decreased processing time, although several technical difficulties remain to be addressed. Immunomagnetic separation offers significant potential for improvement on existing microbiological systems for the isolation of salmonella.

Animals↗
PubMed2000

Immunomagnetic separation of Cryptosporidium parvum oocysts using MACS MicroBeads and high gradient separation columns.

We evaluated the MACS immunomagnetic separation (IMS) system for concentrating Cryptosporidium parvum. Oocysts were first labeled with fluorescein isothiocyanate (FITC) or rabbit anti-C. parvum antibodies, then linked to MicroBeads coated with anti-FITC or anti-rabbit IgG, and separated through a high gradient separation column. Results indicated that over 95% of oocysts were recovered and their fluorescence and infectivity were retained. The presence of MicroBeads showed no effect on genomic DNA extraction and subsequent polymerase chain reaction (PCR)-based analyses, as sensitivity of PCR (10 oocysts) and the band pattern of randomly amplified polymorphic DNA (RAPD) were identical to those using DNAs extracted from normally purified oocysts. IMS-PCR consistently detected as few as 10 oocysts from 100 ml of apple juice or homogenized milk and IMS-IFA could detect 100 oocysts from 1 g of deer manure, demonstrating the efficiency of IMS in recovering oocysts from environmental and food samples. Our results suggest that the MACS IMS system could be used for multiple applications in Cryptosporidium research.

Animals↗
PubMed2002

Detection of Escherichia coli O157:H7 using immunomagnetic separation and absorbance measurement.

An assay system for detection of Escherichia coli O157:H7 was developed based on immunomagnetic separation of the target pathogen from samples and absorbance measurement of p-nitrophenol at 400 nm from p-nitrophenyl phosphate hydrolysis by alkaline phosphatase (EC 3.1.3.1) on the "sandwich" structure complexes (antibodies coated onto micromagnetic beads--E. coli O157:H7-antibodies conjugated with the enzyme) formed on the microbead surface. The effects of immunoreaction time, phosphate buffer concentration, pH and temperature on the immunomagnetic separation of E. coli O157:H7 from samples were determined and the conditions used for the separation were 1-h reaction time, 1.0 x 10(-2) M PBS, pH 8.0 and 33 degrees C in this system. The effects of MgCl(2) concentration, Tris buffer concentration, pH and temperature on the activity of alkaline phosphatase conjugated on the immuno-"sandwich" structure complexes were investigated after immunomagnetic separation of the target pathogen and the conditions used for the enzymatic amplification were 1.0 x 10(-4) M MgCl(2), 1.0 M Tris buffer, pH 8.0, 28 degrees C and 30-min reaction time during the assay. The selectivity of the system was examined and no interference from the other pathogens including Salmonella typhimurium, Campylobacter jejuni and Listeria monocytogenes was observed. Its working range was from 3.2 x 10(2) to 3.2 x 10(4) CFU/ml, and the relative standard deviation was 2.5-9.9%. The total detection time was less than 2 h.

Alkaline Phosphatase↗
PubMed2001

Effects of antibody concentration on the separation of human natural killer cells in a commercial immunomagnetic separation system.

BACKGROUND: The magnetic separation of a cell population based on cell surface markers is a critical step in many biological and clinical laboratories. In this study, the effect of antibody concentration on the separation of human natural killer cells in a commercial, immunomagnetic cell separation system was investigated. METHODS: Specifically, the degree of saturation of antibody binding sites using a two-step antibody sandwich was quantified. The quantification of the first step, a primary anti-CD56-PE antibody, was achieved through fluorescence intensity measurements using a flow cytometer. The quantification of the second step, an anti-PE-microbeads antibody reagent, was achieved through magnetophoretic mobility measurements using cell tracking velocimetry. RESULTS: From the results of these studies, two different labeling protocols were used to separate CD56+ cells from human, peripheral blood by a Miltenyi Biotech MiniMACS cell separation system. The first of these two labeling protocols was based on company recommendations, whereas the second was based on the results of the saturation studies. The results from these studies demonstrate that the magnetophoretic mobility is a function of both primary and secondary antibody concentrations and that mobility does have an effect on the performance of the separation system. CONCLUSIONS: As the mobility increased due to an increase in bound antibodies, the positive cells were almost completely eliminated from the negative eluent. However, with an increase in bound antibodies, and thus mobility, the total amount of positive cells recovered decreases. It is speculated that these cells are irreversibly retained in the column. These results demonstrate the complexity of immunomagnetic cell separation and the need to further optimize the cell separation process.

Antigen-Antibody Reactions↗
PubMed2000

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.

Animals↗