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PubMed2003

An adapted ImmunoMagnetic cell separation method for use in quantification of Escherichia coli O157:H7 from bovine faeces.

Detection of Escherichia coli O157:H7 organisms in food, clinical or environmental samples is necessary for diagnosis of infection and epidemiological investigations. However, this pathogen may be present in low numbers and difficult to identify among high numbers of other background bacteria. In order to increase the sensitivity of culture- and PCR detection, pre-enrichment of E. coli O157:H7 in broth culture combined with ImmunoMagnetic cell Separation (IMS) is routinely employed. These methods, although able to detect levels as low as 2 cfu/g (from 10 to 25 g samples), are qualitative detection strategies only. If the actual numbers of E. coli O157:H7 are to be quantified, growth enrichment must be excluded and the organisms isolated directly from the sample of interest. Such quantification is necessary, for example, to determinate contamination levels on beef carcasses and for determination of bacterial numbers in in vivo gene expression studies. In the present study, it was not possible to recover organisms from bovine faecal suspensions using the customary IMS system and so a range of alternative buffers and other paramagnetic beads was tested. Combination of a 6.2-microm diameter bead with a detergent-based buffer gave optimal recovery of E. coli O157:H7 organisms from faecal suspensions. This system was validated for recovery of E. coli O157:H7 by comparing it with that obtained with the standard Dynabeads IMS protocol, using both the traditional broth enrichment method and a quantitative detection approach. We conclude that a 6.2-microm diameter Aureon bead can be used for quantitative isolation of E. coli O157:H7 directly from bovine faeces and, for this purpose, is preferred to the 2.8-microm diameter Dynal bead.

Animals↗
PubMed1996

Rapid and sensitive detection of Shigella sonnei in feces by the use of an O-antigen-specific monoclonal antibody in a combined immunomagnetic separation-polymerase chain reaction assay.

OBJECTIVE: The aim of the present work was to develop a rapid, specific and highly sensitive diagnostic method for the detection of Shigella sonnei directly from stool samples. DESIGN AND METHODS: An immunomagnetic separation-polymerase chain reaction (IMS-PCR) combined assay for diagnosis of S. sonnei was developed. For this, a monoclonal antibody (Mab) specific for the O-antigen of S. sonnei was coated to magnetic beads for capture, concentration and separation of S. sonnei from feces. Bacterial DNA, was amplified by the PCR with specific primers. The amplified products were developed by dot blot hybridization with a specific alkaline phosphatase-conjugated probe. RESULTS: The purified MASS MAb reacted specifically with the Plesiomonas shigelloides (the same O-antigen as Shigella sonnei) LPS. The primers were specific for invasive Shigella and enteroinvasive Escherichia coli. Only invasive Shigella sonnei strains gave a positive result in the IMS-PCR assay. The detection limit was 10 to 15 c.f.u. CONCLUSIONS: The availability of IMS-PCR assays provides an improved method for the diagnosis of shigellae directly from feces. The assay is rapid, highly sensitive and specific for the detection of Shigella sonnei directly from stool samples.

Journal Article↗
PubMed1997

Detection, distribution and probable fate of Escherichia coli O157 from asymptomatic cattle on a dairy farm.

The use of commercial anti-Escherichia coli O157-labelled magnetic beads was investigated to improve detection of E. coli O157 by immunomagnetic separation (IMS) from a range of environments on a dairy farm. Immunomagnetic separation proved effective for separation of target cells from laboratory mixtures and during stress in sterile and non-sterile pond water. The IMS procedure was possible with a range of samples (water, faeces, slurry, grass and soil). Non-specific binding of non-target bacterial cells proved problematic in a number of sample types. However, indigenous E. coli O157 cells were detected from samples with a high faecal load, and only with use of IMS. Data on the probable survival and spread of the organism around the farm environment are also discussed.

Animals↗
PubMed1998

Purging and haemopoietic progenitor cell selection by CD34+ cell separation.

Tumour cell contamination of autologous peripheral blood stem cell samples (PBSC) and bone marrow (BM) is frequent. Enrichment of CD34+ stem cells is a promising approach to purging tumour cells from autografts without damaging progenitor cells. Breast cancer cells were seeded (10(-3)-10(-7)) into mononuclear cells from G-CSF-mobilised PBSC and BM harvests from patients without breast cancer. CD34+ cells were enriched from mixtures either by immunomagnetic separation (Isolex-50, and MiniMACS) or by biotin-streptavidin immunoaffinity columns (Ceprate-LC). CD34+ cell fractions were determined by FACS, cancer cells were detected immunocytochemically with an anti-pancytokeratin antibody. The CD34+ cells were enriched with a median purity of 92.2% (43.5-96.1) (n = 17) (Isolex-50), 96.5% (66.6-99.2) (n = 17) (MiniMACS) and 77.9% (31.4-93.6) (n = 15) (Ceprate-LC) from PBSC and BM harvests. The percentages of median recovery of CD34+ cells were 30.8% (18.6-71.8) (Isolex-50), 69.9% (39.1-100) (MiniMACS) and 42.9% (23.7-100) (Ceprate-LC). The median tumour cell reductions in log steps were 3.7 (2.9-4.3) (n = 13) (Isolex-50), 3.5 (2.6-4.3) (n = 13) (MiniMACS) and 1.5 (0.9-2.9) (n = 17) (Ceprate-LC). Results were compared statistically by univariate analysis. Purity was significantly (P < 0.05) better after MiniMACS selection. Recovery rates were significantly different between all devices tested. Tumour cell purging was superior after immunomagnetic separation (P < 0.001). Tumour cell purging is a main objective of CD34+ selection in the autologous setting. Our in vitro data clearly indicate that immunomagnetic separation is more efficient in the prevention of accidental reinfusion of contaminating tumour cells compared to immunoaffinity. However, it is not yet known if the same results can be obtained with fresh contaminating tumour cells.

Antigens, CD34↗
PubMed1999

Development and evaluation of an immunomagnetic separation-ELISA for the detection of Staphylococcus aureus thermostable nuclease in composite milk.

An ELISA method based on monodisperse magnetic beads was developed for the detection of Staphylococcus aureus thermostable nuclease (TNase) in composite milk, wherein S. aureus TNase is captured by magnetic beads coated with monoclonal antibodies directed against TNase and subsequently detected by an enzyme-labelled MAb against the same antigen. Sensitivity of the test was approximately 1 ng TNase, which corresponds to the amount of TNase produced and secreted by approximately 10(5) S. aureus per ml. The Immuno Magnetic Separation (IMS)-ELISA detected TNase in samples from which no S. aureus could be demonstrated on culture. The total test time is 3 h and can be performed either on preserved or fresh milk. The method may be automated.

Animals↗
PubMed1988

Monosized, magnetic polymer particles: their use in separation of cells and subcellular components, and in the study of lymphocyte function in vitro.

By employing the principles of "activated swelling", monosized, superparamagnetic polymer particles have been prepared ranging in size from 1-100 microns. Both during and after the swelling process, the particles can be modified to meet a series of specific demands making them potentially very interesting for many separation and assay purposes. Using monoclonal antibodies to direct the magnetic beads to their targets, immunomagnetic separation has turned out to be one of the most specific, reliable and, above all, the fastest technique available today to isolate particulate material for further studies. So far, most efforts have been concentrated on methodology for fractionation of cells in suspension, such as removal of tumour cells from bone marrow or isolation of lymphoid cells from peripheral blood. These studies have both established the parameters necessary for optimal performance and at the same time laid the groundwork for future developments making immunomagnetic separation an exciting new tool in many research areas. High speed and specificity are the most conspicuous features of immunomagnetic cell separation. These properties have been exploited in the successful development of a new technique for tissue typing of cells directly from peripheral blood specimens. Both higher sensitivity and specificity have been obtained. The same principles can be used for fast and safe quantification of cell populations and subpopulations in blood and cell suspensions. The functions of, and interactions between, peripheral blood cell populations or subpopulations in the immune response have also been studied with high precision. The significance of direct cell contact on the one hand, and soluble factors on the other, can now be established in detail. Immunomagnetic beads have also been used to study the interaction between various T lymphocyte membrane molecules in the early phases of the activation process. Finally, the usefulness of specially developed particles for the fractionation of subcellular components is described.

Antibodies, Monoclonal↗
PubMed1997

Expression of functional GABAA receptors in transfected L929 cells isolated by immunomagnetic bead separation.

Transient cotransfection of fibroblasts, with plasmids encoding individual GABAA receptor (GABAAR) subunits, has provided a model to characterize the pharmacological and kinetic properties of receptor subtype combinations. However, identifying transfected cells for electrophysiological recording is often difficult due to low transfection efficiencies. Selection of transfected cells has required cotransfection with a marker gene and fluorescence microscopic localization prior to recording. To circumvent these problems, two GABAAR subtypes combinations in transfected L929 cells were isolated with a novel biomagnetic separation system. Cell selection was accomplished by cotransfection with a plasmid (pHook-1) encoding a single-stranded cell surface antibody (sFv), which bound to ferromagnetic beads, coated with an antigen (phOx). Bead-covered cells were then magnetically separated from non-transfected cells. Bead-selected cells cotransfected with alpha 6, beta 3 and gamma 2L subtypes, expressed GABAAR currents in 95% (41/43) of cells recorded. Cells cotransfected with alpha 5, beta 3 and gamma 2L subtypes had an EC50 for GABA of 5.4 microM and a Hill slope of 1.4. Membrane patches from cells expressing the alpha 5 beta 3 gamma 2L isoform demonstrated single channel currents with a main conductance state of 23 pS. Magnetic bead immunoselection provides a purified population of transfected cells well suited for whole cell and single channel recording.

Animals↗
PubMed1992

Immunomagnetic manipulation of hematopoietic cells: a review of current technology.

During the last decade, immunomagnetic separation of cells has become popular due to the simplicity and speed of the technology. A variety of magnetic materials have been developed that have fundamentally different characteristics. Some of these act as true colloids and have the properties of solutions, while others are larger and are based on the polymerization of materials, such as styrene, in the presence of magnetite. In this article, the different magnetic matrices are discussed and reviewed with respect to their use for the separation of cells of the hematopoietic system. Additionally, a brief introduction into the field of immunomagnetic separation of nucleic acids is presented, as this technology may be used to perform investigations at the molecular level on hematopoietic cells.

Erythrocytes↗
PubMed2005

A study of the prevalence and enumeration of Salmonella enterica in cattle and on carcasses during processing.

Salmonella prevalence and counts were estimated for samples from the oral cavity, hide, rumen, and feces of 100 cattle at slaughter and from the pre- and postchill carcasses of these cattle. Samples were collected from 25 consecutively slaughtered cattle from each of four unrelated groups slaughtered at a single abattoir on different days. Ten additional fecal samples from each group were collected from their respective abattoir holding pens prior to slaughter. The prevalence of Salmonella was estimated using automated immunomagnetic separation, and the counts were estimated using a combination of most probable number (MPN) and automated immunomagnetic separation. A total of 606 samples were collected with Salmonella isolated from 157 (26%), including 29% of oral cavities, 68% of hides, 16% of feces collected after evisceration, 25% of rumen samples, 2% of prechill carcasses, 3% of postchill carcasses, and 48% of feces collected from holding pens. The prevalence and count of Salmonella varied between the different groups of animals tested. The highest count obtained was from a rumen sample (1.1 x 10(4) MPN/g). Other counts were generally low, with a maximum count in feces collected after evisceration and in the abattoir holding pens of 93 and 23 MPN/g, respectively. The highest count on hides, in oral cavities, and on carcasses was 4.8 MPN/cm2, 23 MPN/g, and 0.31 MPN/cm2, respectively. Even though Salmonella was present on the hides and in the rumen and feces of at least one animal from each group of cattle, the processing of animals at this abattoir resulted in few contaminated carcasses, and when contamination occurred, Salmonella was detected at low numbers.

Abattoirs↗
PubMed1998

Isolation of CD34+ cells from blood stem cell components using the Baxter Isolex system.

The CD34 antigen is expressed by human hematopoietic progenitor and stem cells. These cells are capable of reconstituting marrow function after marrow-ablative chemo-radiotherapy. Several different technologies have been developed for the separation of CD34+ cells from bone marrow or peripheral blood stem cell (PBSC) components. We used an immunomagnetic separation technique to enrich CD34+ cells from PBSC components in anticipation of autologous transplantation for patients with B lymphoid malignancies. Twenty-nine patients enrolled on this study and received mobilization chemotherapy followed by G-CSF. Of these, 21 achieved a peripheral blood CD34+ cell level of at least 2.0 x 10(4)/l required by protocol for separation of the stem cell components. A median of three components per patient was collected for processing. The average CD34+ cell concentration in the components after apheresis was 1.0 +/- 1.2%. After the CD34+ cell selection, the enriched components contained 0.6 +/- 0.6% of the starting nucleated cells. The recovery of CD34+ cells, however, averaged 58.4 +/- 19.2% of the starting cell number, with a purity of 90.8 +/- 6.5%. Overall depletion of CD34- cells was 99.96 +/- 0.06%. Nineteen patients were treated with marrow-ablative conditioning regimens and received an average of 6.2 +/- 2.0 x 10(6) CD34+ cells/kg body weight. These patients recovered to an ANC >0.5 x 10(9)/l at a median of 11 days (range 8-14), and platelet transfusion independence at a median of 9 days (range 5-13). Four patients died of transplant-related complications or relapse before 100 days after transplantation. No patient required infusion of unseparated cells because of failure of sustained bone marrow function. These data demonstrate that peripheral blood-derived CD34+ cells enriched by use of an immunomagnetic separation technique are capable of rapid engraftment after autologous transplantation.

Adult↗
PubMed1996

[Verocytotoxin-producing Escherichia coli O157 in Dutch veal calves and beef cattle].

In 1994 (August-September) and in 1995 (July-October) faeces of Dutch veal calves and adult cattle was examined for the presence of verocytotoxin-producing Escherichia coli O157 (O157 VTEC). The samples were collected at slaughterhouses. In 1994, O157 VTEC were isolated from three (0.9%) of 365 fecal samples of veal calves. Faeces of adult cattle was not collected. In 1995, O157 VTEC were isolated from one (0.5%) of 18.3 fecal samples of veal calves and from 30 (11.1%) of 270 fecal samples of adult cattle. In 1994, the organisms were isolated by selective plating onto both sorbitol MacConkey agar (SMAC) and SMAC containing tellurite and cefixime (TC-SMAC) following selective enrichment in modified trypton soya broth with acriflavin (mTSB + a). In 1995, the samples were enriched in modified E. coli broth with novobiocin (mEC + n), and in addition to directly plating onto TC-SMAC the enriched cultures were plated after incorporation of an immunomagnetic separation step of E. coli O157. All 30 strains isolated from adult cattle were isolated with the immunomagnetic separation procedure. Four of the 30 samples were determined positive also by directly plating onto TC-SMAC. The sample from the veal calve was determined positive only by directly plating onto TC-SMAC. Plating onto SMAC resulted only in negative results. Characterization of the O157 VTEC isolates showed similarities between isolates of humans and cattle. Additional experiments need to be done concerning this aspect. From this study it appeared that also Dutch cattle are a reservoir of O157 VTEC.

Animals↗
PubMed2006

Manual exfoliation plus immunomagnetic bead separation as an initial step toward translational research.

CONTEXT: The development of biotechnologic platforms capable of high throughput analysis has ushered in a promising new era of translational medicine. However, most studies to date are based on in vitro cell lines or substitute models for human disease. Although these model systems have proven insightful, it is readily becoming apparent that human clinical tissue must be studied in order to fully understand all the nuances of human disease. Studies that are based on human tissue, however, are limited by qualitative and quantitative issues, factors often precluding their use in high throughput studies. OBJECTIVE: To develop a simple and rapid tissue procurement protocol for use in obtaining a homogeneous epithelial cell population from clinical tissue and the recovery of nucleic acids and proteins of high quality and quantity. Also, to determine if the technique preserves tissue, thereby allowing morphologic correlation with molecular findings. DESIGN: Performance of manual exfoliation to procure cells from clinical resection specimens and use of immunomagnetic beads embedded with the antibody ber-Ep4 for the positive enrichment of a homogeneous epithelial cell population. Nucleic acids and proteins are then separated using a phenol plus guanidine thiocyante solution. Nucleic acids and proteins are quantitated and qualitatively analyzed using standard laboratory techniques. RESULTS: Nucleic acids and proteins of high quality and quantity were recovered following manual exfoliation and immunomagnetic bead separation. Tissue architecture was not destroyed, thus permitting histologic and molecular correlation. CONCLUSIONS: A simple and reproducible protocol is presented that may enable the molecular profiling of clinically resected tissue. Although the technique is currently limited to certain tissue and tumor types, further research will broaden its overall application.

Adenocarcinoma↗
PubMed2005

Detection of Lawsonia intracellularis using immunomagnetic beads and ATP bioluminescence.

Lawsonia intracellularis is an obligate intracellular pathogenic bacterium that causes proliferative enteropathy in various animals. The detection of L. intracellularis in clinical and environmental samples is necessary for the diagnosis of infection and epidemiological investigations. For the detection of L. intracellularis in fecal samples, we have developed an immunological method using immunomagnetic separation and ATP bioluminescence. Magnetic beads were coated with an anti-Lawsonia surface antigen (LsaA) antibody in order to capture the L. intracellularis in fecal samples from infected rabbits and the bacteria captured by the immunomagnetic beads were assayed by means of ATP bioluminescence. Our results showed that L. intracelluraris was detected by immunomagnetic separation of bacteria-holding magnetic beads and ATP-based bioluminescence, suggesting that our methods could be useful for the diagnosis of proliferative enteropathy.

Adenosine Triphosphate↗
PubMed2003

Rainbow trout (Oncorhynchus mykiss) sIgM- leucocytes secrete an interleukin-2 like growth factor after mitogenic stimulation in vitro.

Two secreted proteins were detected in culture supernatants of PHA or PMA stimulated, immunomagnetically separated, sIgM(-) leucocytes of rainbow trout (Oncorhynchus mykiss) with 60kDa and with 12-15kDa (multiple bands). So called conditioned media (CM), containing these proteins, induced significant activation of blood and head kidney leucocytes. Immunomagnetically separated, naive as well as PHA activated sIgM(-) T lymphocytes and LPS prestimulated sIgM(+) B lymphocytes could be identified to be responding to these secreted proteins. Using a monoclonal antibody specific for mouse IL-2 (clone JES6-1A12), one of the multiple 12-15kDa proteins could be stained in Western blots. It was also shown that the induced proliferation was due to this protein in the CM, as the same anti-IL-2 mab was able to block the CM induced proliferation. Furthermore, survival of the IL-2 dependent mouse cell line HT-2 was enhanced after addition of various concentrations of CM. The data presented show, for the first time, that mitogen stimulated trout sIgM(-) leucocytes secrete a cytokine like growth factor sharing functional and structural similarities with mammalian IL-2.

Animals↗
PubMed1993

Immunomagnetic positive selection and colony culture of CD34+ cells from blood.

Immunomagnetic separation has been used to enrich CD34-positive cells in umbilical cord blood. Cell purities were increased from 0.59% preseparation to 92.7% postseparation (n = 16) with a mean yield of 75.7%. CFU were enriched 127 fold by immunomagnetic separation. Addition of combinations of recombinant growth factors resulted in cloning efficiencies of greater than 50%.

Antibodies, Monoclonal↗
PubMed1991

Negative selection of human monocytes using magnetic particles covered by anti-lymphocyte antibodies.

We have developed a standardized procedure for the isolation of monocytes from peripheral blood by negative selection using magnetic polymer particles coated with monoclonal antibodies against T and B lymphocytes. The average purity of the monocyte suspension was 85%, and monocyte recovery was 72% from Ficoll-Hypaque gradient separated mononuclear cells and 32% from whole blood. In a lucigenin enhanced chemiluminescence assay there was no significant difference between cells separated immunomagnetically and those separated on a gradient. Nor did electron microscopy show any significant difference in morphology between such monocytes. Negative selection using magnetic polymer particles is an efficient method for the separation of monocytes with intact morphology and function as measured by chemiluminescence.

Antibodies, Monoclonal↗