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Results for “Immunomagnetic Separation”

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PubMed2005

The detection of Cryptosporidium parvum and Escherichia coli O157 in UK bivalve shellfish.

Optimised immunomagnetic separation methods to detect Cryptosporidium parvum and Escherichia coli O157 in UK shellfish are described. Whole tissue homogenates gave the best recoveries for C. parvum oocysts compared with gill or haemolymph extracts. The sensitivity of recovery from spiked samples was comparable to that achieved when processing water and varied from 12-34% in mussels, 48-69.5% in oysters and 30-65% in scallops. Maximum recovery of E. coli O157 was achieved by enriching in buffered peptone water supplemented with vancomycin at 42 degrees C. Increasing enrichment temperatures from 37 to 42 degrees C gave a significant increase in target number recovery. Implementation of these methods into monitoring programmes and end-product testing will enable shellfish producers to better assess product safety.

Animals↗

PubMed2000

Cattle can be a reservoir of sorbitol-fermenting shiga toxin-producing Escherichia coli O157:H(-) strains and a source of human diseases.

Using the immunomagnetic separation procedure, we isolated sorbitol-fermenting (SF) Shiga toxin-producing Escherichia coli (STEC) O157:H(-) strains from two patients, one with hemolytic-uremic syndrome and the other with diarrhea, and from a dairy cow epidemiologically associated with the patients. The phenotypic and genotypic characteristics of all isolates were identical or closely related. Moreover, the bovine isolate showed a clonal relatedness to SF STEC O157:H(-) strains isolated from patients in Germany and the Czech Republic from 1988 to 1998. This is the first evidence that cattle can be a reservoir of SF STEC O157:H(-) and a source of human diseases.

Animals↗

PubMed2002

Synthesis of a group-selective antibody library against haptens.

A generic strategy is described for the generation of libraries comprising hapten-selective antibody genes against a group of structurally related low molecular weight target molecules. Hapten antibody libraries are frequently suffering from high background levels of irrelevant antibody genes as a consequence of the immunization, where the small non-immunogenic target molecule is coupled to a large immunogenic carrier protein. In order to elevate the percentage of hapten-specific genes in the library, B cells harboring antibody genes against the group of triazine herbicides were enriched from 21 individual splenocyte populations by means of immunomagnetic separation (IMS). IMS utilizes the specific binding of membrane-associated immunoglobulin receptors on the B cell surface to hapten-coated paramagnetic beads. The variable genes of the specifically enriched subpopulation were cloned into a phagemid vector. The corresponding library yielded up to 75% triazine binding antibody clones after three rounds of phage selection. At least half of these antibodies (abs) were displaceable by triazines resulting in quantitative assays with nanomolar sensitivities. In contrast, no displaceable clone was obtained at the same selection level in a control library, where IMS was omitted. Due to the elevated percentage of relevant antibody genes, the library can be utilized either for the direct isolation of functional antibodies against various triazine herbicides or as group-specific gene source for evolutionary antibody optimization.

Animals↗

PubMed1995

Vascular cell adhesion molecule 1-positive reticular cells express interleukin-7 and stem cell factor in the bone marrow.

In vitro studies have defined an essential role for stromal cells in supporting B-cell development, including production of lymphopoietic cytokines. It has been suggested that stromal cells are equivalent to adventitial reticular cells in the marrow; however, evidence of reticular cells producing cytokines has been difficult to obtain. Staining of bone marrow (BM) sections with antibodies to interleukin-7 (IL-7) showed a reticular pattern, mimicking that obtained using antibodies to vascular cell adhesion molecule 1 (VCAM-1), a molecule present on both stromal cells in vitro and reticular cells. To more closely examine cytokine production within normal marrow, an immunomagnetic separation scheme was devised to directly enrich VCAM-1+ stromal cells. Twenty to thirty percent of cells isolated in the VCAM-1+ fraction shared characteristics with stromal cells from long term BM cultures, including cellular morphology and expression of alkaline phosphatase and alpha actin. These were termed "reticular stromal" cells. Immunohistochemical staining showed that virtually all of the latter cells possessed cytoplasmic IL-7 protein, and about half expressed stem cell factor. In contrast with cultured stromal cells, very few had detectable macrophage-colony-stimulating factor. These data constitute the first report of cytokine expression by marrow reticular cells in vivo. The implications of this data with respect to the existence of stromal cell subsets and their regulation of lymphopoiesis is discussed.

Actins↗

PubMed1998

H-Y antigen expression patterns in human X- and Y-chromosome-bearing spermatozoa.

PROBLEM: Restricted expression of H-Y antigen on Y-chromosome-bearing sperm has been reported in some species, although such preferential expression for H-Y antigen in human sperm has yet to be described. In this study, an immunomagnetic approach was used to characterize antigen expression patterns as a function of sex-chromosome content. METHOD OF STUDY: Human sperm was treated with monoclonal immunoglobulin (Ig) M antibodies directed against H-Y antigen. This preparation then was incubated with sheep antimouse IgM antibody affixed to paramagnetic beads, which then were exposed to a magnetic field and sorted. X- and Y-chromosome frequencies in the two subgroups of sperm were assayed by multiprobe fluorescent in situ hybridization (FISH). RESULTS: Sperm were immunomagnetically separated into two populations: a reactive group (presumably, H-Y Ag+); and a nonreactive group (presumably, H-Y Ag-). Triple-color FISH analysis of 1,600 spermatozoa (800 in each group) showed the antigen's expression to be somewhat more prevalent among Y-chromosome-bearing sperm (54.1%), but a large proportion of Y-chromosome-bearing sperm (49.0%) did not express this antigen. The difference was not significant (P = 0.43). CONCLUSIONS: The expression of H-Y antigen has a slightly higher frequency in human sperm containing the Y-chromosome, but its expression among X-chromosome-bearing sperm also is considerable. Current immunologic techniques relying on this antigen are unlikely to effect the sex selection of human sperm.

Animals↗

PubMed1998

Luteal leukocytes are modulators of the steroidogenic process of human mid-luteal cells.

Flow cytometry analysis of luteal cells revealed that an important proportion of these cells are leukocytes. The percentage of leukocytes was higher in the early (42 +/- 4) and late (35 +/- 3) luteal phases than in the mid-luteal (24 +/- 2) phase. However, the proportion of macrophages did not differ between the luteal stages. The flow cytometric properties correlated with cellular size and granularity were not reliable as discriminators of luteal cell subpopulations. Therefore, to assess the contribution of luteal leukocytes, these cells were completely removed from luteal cell suspensions (total cells), by a negative selection procedure (immunomagnetic separation). The functional role of leukocytes in mid-luteal steroidogenesis was assessed, in total as well as leukocyte-depleted cells. Progesterone production was found to have increased 2.2-fold in leukocyte-depleted cell cultures, in comparison with total cells under basal conditions. However, the response to human chorionic gonadotrophin (HCG) was 36% lower under the latter conditions. Oestradiol production was not significantly modified under basal or HCG-treated conditions. In leukocyte-depleted cells, the concentration of interleukin (IL)-1beta decreased 5-fold in comparison with total cell cultures, suggesting that leukocytes are the principal source of IL-1beta. In summary, the results of the present investigation suggest functional interactions between the immune system and steroidogenic cells of the human corpus luteum.

Adult↗

PubMed2002

Detection of eight Cryptosporidium genotypes in surface and waste waters in Europe.

Cryptosporidia are important protozoan parasites of vertebrates, and a number of species and genotypes, with different host ranges, have been described. In this study a protocol was established for the detection and the genetic characterization of Cryptosporidium spp. isolated from various types of surface waters (rivers, creeks, lakes, sewage plant in- and outlets and swimming pools) from the area between Zurich (Switzerland) and Munich (Germany). Cryptosporidium oocysts were isolated by continuous-flow-centrifugation and immunomagnetic separation (IMS). A novel nested PCR combined with direct sequencing of the amplicon which spans a variable region of the 18S rRNA allowed characterization of species and genotypes. Cryptosporidium spp. were detected in 23 of the 68 water samples investigated. Almost half of these isolates represent species and genotypes known to be pathogenic to man, namely C. parvum 'bovine genotype' (from 6 samples) and C. parvum 'human genotype' (4). Furthermore, we identified C. muris 'genotype A' (3), C. muris 'genotype B' (6), C. baileyi (1) as well as 3 novel Cryptosporidium genotypes. Our results confirm the ubiquitous presence of Cryptosporidium oocysts in surface waters. The detection of a variety of species and genotypes stresses the importance that molecular characterization is indispensable before drawing conclusions of medical or epidemiological significance.

Animals↗

PubMed2000

Rapid detection of Salmonella typhimurium in chicken carcass wash water using an immunoelectrochemical method.

An immunoelectrochemical method coupled with immunomagnetic separation was developed for rapid detection of Salmonella Typhimurium in chicken carcass wash water. Samples of chicken carcass wash water were inoculated with Salmonella Typhimurium at different cell numbers. Possible nonspecified inhibitors in the wash water were minimized by filtration and centrifugation. An approximately 9.4% loss of Salmonella cells was found after filtration (P < 0.01). The samples were mixed with anti-Salmonella-coated magnetic beads (ASCMB) and alkaline phosphatase-labeled anti-Salmonella (APLAS) to form ASCMB-Salmonella-APLAS conjugates. The conjugates were separated from the solution using a magnetic separator and then incubated with phenylphosphate substrate to produce phenol. The number of Salmonella was determined by measuring the phenol concentration using an amperometric tyrosinase carbon paste electrode in a flow injection analysis system. Under optimized parameters (1 mM MgCl2, 0.2 microg/ml APLAS, and 1 mM phenylphosphate in pH 7.0 Tris buffer solution), Salmonella Typhimurium in chicken carcass wash water could be identified and enumerated within 2.5 h with a detection limit of 5 x 10(3) CFU/ml. A linear relationship on a log-log scale was found between Salmonella cell number and the peak current ratio for Salmonella concentrations ranging from 10(3) to 10(7) CFU/ml (R2 = 0.963). The peak currents of multibacteria samples, containing Salmonella Typhimurium, Listeria monocytogenes, and Campylobacter jejuni, were not significantly different from Salmonella-only samples (P > 0.01).

Animals↗

PubMed2003

Phenotypic and molecular characteristics of typical and atypical Escherichia coli O157, clinical and food isolates.

Enrichment, colony isolation and confirmation are three general phases of a standard diagnostic method. E. coli O 157 (the main member of EHEC group) differs metabolically from other strains of E. coli in a number of ways. Most isolates are slow- or non-fermenters of sorbitol and lack the enzyme beta-glucuronidase (GUD). But, a variety of atypical strains of E. coli O157 (sorbitol-fermenting variants, nonmotile and GUD-positive) have been reported. The discovery of these atypical pathogenic strains brings into question the validity of testing for the pathogen only by biotyping. Using classical cultivation and immunomagnetic separation, we have isolated from food a few atypical E. coli O157 (sorbitol-fermenting strains, GUD positive, nonmotile O157 strain which does not agglutinate with O157 latex and does not produce Shiga toxin). On the other hand, non-O157 VTEC (O26 serotype) producing Shiga toxin was isolated from meat. Molecular markers of E. coli O157 and virulence-associated factors of strains with aberrant biochemical properties were studied by PCR. This method helped us in the final identification of isolates. Since it was suggested that the production of verotoxins (VT) is accompanied by the production of enterohemolysin (Ehly) such correlation has also been evaluated in respect to the collection of VTEC of human, animal and food origin.

Animals↗

PubMed2005

Noninvasive prenatal diagnosis for hemoglobin Bart's hydrops fetalis.

Hemoglobin (Hb) Bart's hydrops fetalis, the most severe thalassemic disease, occurs from homozygosity of alpha-thalassemia 1. Deletion of all 4 alpha-globin genes (- -/- -) in this condition results in the absence of alpha-globin chains, and the physiologic dysfunction of Hb Bart's (gamma4) leads to lethality, either in utero or soon after birth. The best strategy for prevention and control of the disease is prenatal diagnosis in the mothers at risk. However, conventional prenatal diagnosis involves invasive procedures that may result in infection or abortion. In this study, a simple technique was developed to identify the presence or absence of alpha-globin chains in fetal nucleated red blood cells (NRBCs) enriched from maternal blood. Mononuclear cells including fetal NRBCs were isolated from maternal blood at 10 to 26 weeks of pregnancy by density-gradient centrifugation. Immunomagnetic separation with anti-CD71 antibody was employed to enrich fetal NRBCs, whose numbers increase with increasing gestational age. For the unaffected fetus, fetal NRBCs were detected by immunofluorescence microscopy after staining with rabbit antihuman alpha-globin antibody. In contrast, fetal red blood cells homozygous for alpha-thalassemia 1, which were identified from their size and morphology, did not stain for alpha-globin antibody. In this study, 3 affected fetuses were detected from 10 pregnancies at risk of Hb Bart's hydrops fetalis, and the results were confirmed by DNA analysis. In the remaining cases, all fetal NRBCs were positive for immunofluorescence staining. DNA analysis revealed that 2 cases were normal, 1 was heterozygous for alpha-thalassemia 2, and the other 4 cases were heterozygous for alpha-thalassemia 1.

Erythroblasts↗

PubMed2005

Partial hepatectomy induces mobilization of a unique population of haematopoietic progenitor cells in human healthy liver donors.

BACKGROUND/AIMS: Recent studies indicate that after transplantation, circulating bone marrow-derived stem cells migrate into the liver and contribute to liver regeneration. Whether such cells are actively recruited from the bone marrow for liver repair remains to be determined. In this regard, we investigated whether liver resection leads to a release of stem cell marker-positive (+) cells into the peripheral circulation. METHODS: Peripheral blood samples from 11 living liver donors were analyzed by flow cytometry one day before and 12h after partial hepatectomy (PH) using antibodies against CD133, CD34, CD45, CD14, c-kit, bcrp-1. Immunomagnetic separation was performed to select CD133+ cells for functional assays in vitro. RESULTS: A significant increase in the percentage of CD133+ cells could be demonstrated in all donors studied. Unexpectedly, virtually all CD133+ cells coexpressed CD45 and CD14, whereas only a small subset expressed CD34. No significant staining was observed for c-kit and bcrp-1. In culture, immunoselected CD133+ cells displayed characteristics of myelomonocytic precursors. In addition, enriched CD133+ generated an adherent cell population that expressed CK8, alpha-fetoprotein, and human albumin. CONCLUSIONS: PH induces mobilization of a distinct population of myelomonocytic progenitor cells, which have hepatic differentiation potential in vitro, and might play a role in liver regeneration after PH in humans.

Antigens, CD↗

PubMed2006

Cryptosporidium oocysts in mussels (Mytilus edulis) from Normandy (France).

Cultured mussels (Mytilus edulis) were collected seasonally during one year from three sites on the Northwestern coastal area of Normandy (France). Flesh, gills and innerwater were examined for Cryptosporidium oocyst detection using immunomagnetic separation and immunofluorescence assay. Oocysts were present in all samples for all sites and seasons and flesh was the most contaminated part. Oocyst rates were apparently related with seasonal rain precipitation variations. Molecular analysis revealed that oocysts belonged to the species Cryptosporidium parvum (formerly genotype 2 or < >). Oocyst infectivity was assessed by oral administration to suckling NMRI-mice, and developmental stages were observed in only one mouse infected with oocysts from one location. The detection of potentially infectious C. parvum oocysts of likely cattle-breeding origin in cultured edible mussels confirms their resistance to sea environments, and underlines the potential risk of food-borne infection. This work reports for the first time the presence of infectious Cryptosporidium oocysts in shellfish from France.

Animals↗

PubMed2005

Genotypes of Cryptosporidium from Sydney water catchment areas.

AIMS: Currently cryptosporidiosis represents the major public health concern of water utilities in developed nations and increasingly, new species and genotypes of Cryptosporidium are being identified in which the infectivity for humans is not clear. The complicated epidemiology of Cryptosporidium and the fact that the majority of species and genotypes of Cryptosporidium cannot be distinguished morphologically makes the assessment of public health risk difficult if oocysts are detected in the raw water supplies. The aim of this study was to use molecular tools to identify sources of Cryptosporidium from the Warragamba catchment area of Sydney, Australia. METHODS AND RESULTS: Both faecal and water samples from the catchment area were collected and screened using immunomagnetic separation (IMS) and immunofluorescence microscopy. Samples that contained Cryptosporidium oocysts were genotyped using sequence and phylogenetic analysis of the 18S rDNA, and the heat-shock (HSP-70) gene. Analysis identified five Cryptosporidium species/genotypes including C. parvum (cattle genotype), C. suis, pig genotype II, the cervid genotype and a novel goat genotype. CONCLUSIONS: Monitoring and characterization of the sources of oocyst contamination in watersheds will aid in the development and implementation of the most appropriate watershed management policies to protect the public from the risks of waterborne Cryptosporidium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that quantification by IMS analysis can be combined with the specificity of genotyping to provide an extremely valuable tool for assessing the human health risks from land use activities in drinking water catchments.

Animals↗

PubMed2002

Colony-blot assay with anti-p60 antibodies as a method for quick identification of Listeria in food.

The present study evaluated the ability to isolate Listeria from foods, using shortened procedure of sample enrichment followed by immunomagnetic separation or filtration methods, and serological identification of isolated bacteria by colony-blot and Western blot methods with anti-p60 antibodies. By these rapid methods, identification of Listeria was achieved in much shorter time (40-48 h) than with standard cultivation and biochemical identification procedures. The rapid methods used are easy to perform and, what is most important, their specificity is very high and fulfills the expectations. The possibility to select Listeria colonies growing on non-selective media by blotting with anti-p60 antiserum seems to be particularly valuable in examination of food samples containing/not too many Listeria (1-10 CFU/25 g). However, the blot method using anti-PepD mAb specific to unique region of L. monocytogenes p60 is necessary to distinguish L. monocytogenes from other Listeria species.

Antibodies, Bacterial↗

PubMed1998

Dynabeads plus 3 M Petrifilm HEC versus Vitek Immunodiagnostic Assay System for detection of E. coli O157 in minced meat.

The potentially low infective dose of Escherichia coli O157 makes it necessary to be able to detect low numbers in food, and the lack of sensitivity of direct plating has led to the development of various enrichment and detection methods. Until now, the most selective procedure for detection of E. coli O157 isolates was the immunomagnetic separation (IMS) method. The number of sorbitol non-fermenting micro-organisms other than E. coli O157 that adhere non-specifically to the magnetic beads hampers the application of IMS. The use of IMS in conjunction with 3 M Petrifilm-HEC yielded EHEC O157 in 21 of 165 samples of minced meat (12.7%). Without advance application of IMS, Petrifilm plates often yield confluent growth and colonies too numerous to count. The Vitek Immunodiagnostic Assay System (VIDAS-ECO) showed good sensitivity when testing artificially contaminated beef samples, but only four of 21 naturally contaminated samples were recognized. The addition of 3 M Petrifilm to IMS resulted in less growth of contaminants and eliminated much of the need to test presumed colonies for confirmation. The combination of IMS and 3 M Petrifilm-HEC is a fast and efficient screening procedure for E. coli O157 in minced meat.

Animals↗

PubMed2001

Persistence of verocytotoxin-producing Escherichia coli O157:H7 in calves kept on pasture and in calves kept indoors during the summer months in a Swedish dairy herd.

In 1997, a Swedish dairy farm was implicated in a human case of verocytotoxigenic Escherichia coli (VTEC) infection. The bacterium was found in a faecal sample from the human case and in faecal samples from cattle on the farm. Subtyping with pulsed field gel electrophoresis (PFGE) showed that the isolates were identical. The farm was further studied to assess the occurrence and the epidemiology of the agent at the farm level. The objective of this part of the study presented here was to examine the persistence of VTEC O157:H7 in calves that were kept on pasture and indoors, respectively, during the summer. Twelve calves in the herd, with one positive faecal sample each of VTEC O157:H7 in April 1999, were followed by faecal sampling during the summer months. Six calves were kept indoors and six were kept on pasture. Faecal samples from each calf were collected once a month on five occasions from April to September. Bacterial examination was performed with immunomagnetic separation (IMS) and cultivation on CT-SMAC. PCR was used to test for the presence of genes encoding for verocytotoxin (VT), intimin (eaeA), enterohemorrhagic E. coli-hemolysin (EHEC-Hly) and the flagellar antigen H7. PFGE was used for genotyping the isolates. The faecal samples from the calves kept on pasture were negative during the whole period. It is possible that the faecal samples had bacterial counts lower than the detection limits for our procedure, or that the faecal samples were free from the bacteria at the time of sampling. This suggests that calves on pasture may be less exposed to the bacteria or that they clear themselves. In the pen group, there were between one and six culture positive individuals per sampling occasion. One of the calves that was housed indoors was positive in faecal culture on four consecutive samplings.

Animals↗

PubMed1994

Quantitation by competitive PCR of HIV-1 proviral DNA in epidermal Langerhans cells of HIV-infected patients.

Langerhans cells (LC) belong to the dendritic cell family and represent the principal antigen presenting cells populating squamous epithelia. We have reported the presence of human immunodeficiency virus Type 1 (HIV-1) proviral DNA and RNA in purified LC from the epidermis of seropositive patients. The aim of this study was to quantify HIV-1 proviral DNA in LC of infected patients using a competitive polymerase chain reaction (PCR) assay. Bulk epidermal cell (EC) suspensions were obtained from the skin of nine AIDS patients and six seronegative subjects. Purified LC and LC-depleted EC were prepared by immunomagnetic separation using an anti-CD1a monoclonal antibody. LC preparations did not contain T cells, as assessed by reverse transcription PCR analysis of the T cell receptor beta-chain gene (C region). In addition, no CD14+ cells could be detected in LC fractions by immunostaining of cytospin preparations. To quantify HIV-1 DNA, a new competitive PCR system was devised using SK145/150 as primers (gag) and a competitor plasmid DNA with a modified sequence (209 instead of 142 bp). The number of HIV-1 DNA copies found in the LC of AIDS patients ranged from 107 to 3,645/10(5) LC. In contrast, LC-depleted EC from the same subjects were all negative. The results indicate that in AIDS patients the frequency of infected LC is comparable to that reported for peripheral blood CD4+ T cells.

Acquired Immunodeficiency Syndrome↗

PubMed2005

Clams (Corbicula fluminea) as bioindicators of fecal contamination with Cryptosporidium and Giardia spp. in freshwater ecosystems in California.

This study evaluated clams as bioindicators of fecal protozoan contamination using three approaches: (i) clam tissue spiking experiments to compare several detection techniques; (ii) clam tank exposure experiments to evaluate clams that had filtered Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions; (iii) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. Our spiking and tank experiments showed that direct fluorescent antibody (DFA), immunomagnetic separation (IMS) in combination with DFA, and PCR techniques could be used to detect Cryptosporidium in clam tissues. The most analytically sensitive technique was IMS concentration with DFA detection of oocysts in clam digestive gland tissues, which detected 10 oocysts spiked into a clam digestive gland 83% of the time. In the tank experiment, oocyst dose and clam collection time were significant predictors for detecting Cryptosporidium parvum oocysts in clams. In the wild clam study, Cryptosporidium and Giardia were detected in clams from all three study regions by IMS-DFA analysis of clam digestive glands, with significant variation by sampling year and season. The presence of C. parvum DNA in clams from riverine ecosystems was confirmed with PCR and DNA sequence analysis.

Animals↗