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PubMed2000

Detection and speciation of Cryptosporidium spp. in environmental water samples by immunomagnetic separation, PCR and endonuclease restriction.

Current methods for the detection of Cryptosporidium oocysts in water samples are both time-consuming and subject to variation in sensitivity. A genus-specific PCR assay was designed for the specific amplification of a 552-bp region of the 18S rRNA gene. Postamplification endonuclease restriction generated unique digest patterns that enabled differentiation between the three species, C. muris, C. baileyi and C. parvum, the major human pathogen. Theoretical restriction profiles for other Cryptosporidium species were also predicted. The assay routinely detected 10 oocysts in 10-ml purified oocyst preparations, but sensitivity was found to be 10(3)-10(4) -fold lower in environmental water samples. The use of Chelex resin and an immunomagnetic separation procedure overcame this inhibition. This provided detection levels of 10(1)-10(3) oocysts, depending on water turbidity. Rapid and sensitive pathogen detection methods are essential for the water industry. The results of this study demonstrate that PCR has the potential to improve current detection capabilities greatly by differentiating the major human pathogens from non-pathogenic species. This will greatly facilitate a closer examination of the epidemiology of this important pathogen.

Animals↗
PubMed2006

Detection of viable enterohemorrhagic Escherichia coli O157 using the combination of immunomagnetic separation with the reverse transcription multiplex TaqMan PCR system in food and stool samples.

Enterohemorrhagic Escherichia coli O157:H7 is an infectious pathogen and outbreaks have been reported all over the world, specifically in Australia, Canada, Japan, the United States, and in various countries in Europe and South Africa. Therefore, it is necessary to develop rapid methods to determine the target pathogens for food sanitation and disease. Three combinations of primers and probes were designed to detect and identify E. coli O157 using the TaqMan detection system which focuses on the specific genes eae, rfb(O157, and stxII. Reverse transcription (RT) multiplex TaqMan PCR was carried out to accurately detect viable target cells correctly. Furthermore, the acidic pretreatment and immunomagnetic separation (IMS) of food and stool samples also improved the specificity and accuracy of the RT multiplex TaqMan PCR. The developed multiplex TaqMan PCR was effective in differentiating E. coli O157, enterovirulent E. coli, and non-E. coli pathogens from 100 strains which were isolated from clinical patients and the environment. Viable and nonviable cells were also distinguished by this assay. The pretreatment protocol, which included IMS to concentrate and purify the E. coli O157, was developed and the sensitivity of the assay was improved to 10(0) CFU/ml in pure culture, food, and stool samples. The TaqMan PCR assay is a rapid test for the detection of E. coli O157 in food and stool matrices. It shortens the process time and increases the specificity of the pathogens detected. This is critical for improving the safety and sanitation of our food supply.

Adhesins, Bacterial↗
PubMed2005

Combined use of an immunomagnetic separation method and immunoblotting for the enumeration and isolation of Escherichia coli O157 in wastewaters.

AIMS: The detection of Escherichia coli O157:H7 in environmental samples is a human concern. The high persistence of this serotype in the environment suggests that contaminated animal wastewater could act as a potential reservoir. Nevertheless, the high levels of background microflora and cell damage because of environmental stress hamper the isolation of this pathogen without using enrichment methods. This study develops a method for the detection of E. coli and investigates its prevalence in animal and human wastewaters. METHODS AND RESULTS: Incubation of the sample for 1 h 30 min at 37 degrees C in peptone water supplemented with vancomycin and cefsulodin, enhanced the recovery of bacteria whilst ensuring that no growth occurred. Subsequently, a combination of immunomagnetic separation, cefixime-tellurite-sorbitol MacConkey (CT-SMAC) plating and immunoblotting with specific O157 antibodies allowed the detection, enumeration and isolation of E. coli O157 strains in human, swine and cattle wastewaters, which presented values of 0.2, 0.4, and 1.0 log10 ml(-1) units, respectively. Some of the isolates carried genes coding for Shiga toxins, intimin and enterohemolysin. CONCLUSIONS: Escherichia coli O157 is commonly present in animal and human wastewaters. The developed method reduced the high rate of false positives reported for other technical approaches. SIGNIFICANCE AND IMPACT OF THE STUDY: The confirmation of serotype by specific immunomethods is necessary to prevent false-positive detection and incorrect enumeration.

Animal Husbandry↗
PubMed1995

Immunomagnetic separation and PCR for detection of Helicobacter pylori in water and stool specimens.

The detection of Helicobacter pylori in clinical and environmental samples by PCR sometimes requires removal of polymerase inhibitors. We have used a magnetic immunoseparation technique as pre-PCR treatment to facilitate direct detection of H. pylori in stool and water specimens. Rabbit hyperimmune antiserum was produced and magnetic beads were coated with purified immunoglobulin G, which reacted with and bound to both coccoid and rod-shaped forms of H. pylori. When PCR was applied for the detection of H. pylori from cultured samples, the number of organisms that was required for positive scores varied significantly. For a 3-day culture of H. pylori, samples containing 10(2) bacteria per ml are needed for a positive score; for a 6-day culture, samples containing 10(4) bacteria per ml are needed; and for a 10-day culture, samples containing 10(6) bacteria per ml are needed. These results indicate that the coccoid forms of H. pylori may have a different antigenicity and DNA content and are therefore more difficult to detect by immunomagnetic separation and PCR than the rod-shaped forms. Spiked samples with the addition of feces, spiked water samples, and a patient stool specimen were all scored positive with this technique.

Animals↗
PubMed1989

Immunomagnetic separation of infiltrating T lymphocytes from brain tumors.

Tumor-infiltrating lymphocytes (TIL's) were isolated from human glioma biopsy specimens by immunomagnetic separation using T cell-specific monoclonal antibodies coupled to paramagnetic beads, and were expanded in culture with feeder cells and interleukin-2 (IL-2). The infiltrating cells from five of seven patients proliferated in culture. When tested after 2 to 3 weeks of culture, virtually all of the cells stained with antibodies against the CD2 and CD3 antigens. Most cells also expressed human leukocyte antigen class II molecules, while varying percentages of cells stained with antibodies against the IL-2 receptor and the CD4 and CD8 antigens. The cytotoxicity of the cultured TIL's against autologous and allogeneic glioma cells and the K562 and Daudi cell lines was measured and compared with that of lymphokine-activated killer (LAK) cells from the same patients. None of the TIL's showed significant cytotoxicity against these targets, whereas LAK cells lysed all of the targets.

Adult↗
PubMed1991

Effects of target antigen density on the efficacy of immunomagnetic cell separation.

Immunomagnetic cell separation uses binding of an antibody to its epitope to identify the target cell, which is then removed by attachment to an anti-immunoglobulin-coated paramagnetic bead, and passage through a magnetic field. This method has previously been shown to be less sensitive to the effects of low target antigen density than are other cell elimination methods, such as complement-mediated lysis. In this paper we demonstrate that, with certain antibody/target cell combinations, the efficiency of immunomagnetic depletion can be adversely affected by high expression of the target antigen. This can occur by two non-mutually exclusive mechanisms. These are (i) steric hindrance of bead binding due to crowding of monoclonal antibodies on the cell surface; and (ii) binding of the monoclonal antibody molecule in a configuration that is poorly-accessible to the anti-immunoglobulin immobilized on the microspheres. The predominant effect operating in any system can be determined by analysis of the cells remaining after the separation procedure. In both cases pre-attachment of the monoclonal to the beads results in improved separation efficiency. These results emphasize the necessity of optimizing experimental conditions in each system that is investigated.

Antibodies, Monoclonal↗
PubMed1994

An immunomagnetic separation method using superparamagnetic (MACS) beads for large-scale purification of human mammary luminal and myoepithelial cells.

A comparison has been made between different immunomagnetic techniques for separating normal human mammary epithelial cells based on the exclusive expression of EMA (epithelial membrane antigen) by luminal cells and CALLA (CD10) by myoepithelial cells. When cells labelled with antibodies to these antigens were incubated with Dynabeads, they rosetted myoepithelial but not luminal cells. However, both luminal and myoepithelial cells could be positively separated using the MACS system. Purity was established by analyzing the expression of CALLA and EMA using flow cytometry, and of cell-type specific cytokeratins using indirect immunofluorescence. Dynabead-separated myoepithelial cell populations were of high purity (> 98%) but the beads could not be removed from the cells. Luminal cell populations separated by the MACS method were also highly purified (> 95%), as were myoepithelial cell populations (> 90%). Using this immunomagnetic separation method, up to 10(7) cells of each type could be obtained from individual preparations.

Adolescent↗
PubMedDate not supplied

Immunomagnetic separation of antibody-labelled from antibody-free human spermatozoa as a treatment for immunologic infertility. A preliminary report.

A method is described where superparamagnetic polymer microspheres coated with monoclonal antibodies are used to isolate antibody-labelled from antibody-free spermatozoa in male autoimmune infertility. Autoimmune sperm samples or antibody-free spermatozoa adsorbed with antisperm-antibodies from sera were incubated with microspheres coated with a specific monoclonal antibody to murine immunoglobulins, after their preincubation with mouse anti-human IgG and IgA. Using a magnet, the microsphere-labelled spermatozoa were separated from the samples. Immunobead binding was performed before and after the treatment in order to detect changes in the percentage of antibody-bound spermatozoa. After the immunomagnetic separation, approximately 50% of the IgA-labelled spermatozoa was isolated while no difference was demonstrated when antisperm antibodies of IgG class were involved. The evaluation of sperm motility and membrane integrity after treatment seemed to indicate that the technique did not have any relevant effect on sperm characteristics. The fact that only a partial success in separation of IgA-bound spermatozoa and no success for IgG-labelled sperm was obtained indicates that the method needs to be improved before its clinical utilization might be postulated.

Antibodies, Monoclonal↗
PubMed1993

Immunomagnetic separation as a final purification step of liver endothelial cells.

We describe a fast and reproducible method that can be used as a final step in obtaining pure populations of liver endothelial cells. This method employs endothelial cell specific lectin covalently bound to magnetic polystyrene beads (Dynabeads). Evonymus europaeus agglutinin (EEA)-coated Dynabeads were used to purify monkey liver endothelium from Percoll gradient separated nonparenchymal cells. EEA-coated beads were also successfully used to purify monkey aortic endothelial cells. The endothelial cells grew to confluence as a cobblestonelike monolayer, expressed Factor VIII related antigen, and incorporated acetylated-low density lipoprotein. The magnetic beads seemed not to modify the normal properties of the isolated endothelium, thus facilitating their use in experimental studies. This immunomagnetic separation technique may be applicable for purification of endothelial cells from a wide variety of tissue sources.

Animals↗
PubMed1992

Isolation of salmonellas by immunomagnetic separation.

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

Culture Media↗
PubMed1992

Immunomagnetic separation of tumor necrosis factor alpha. I. Batch procedure for human temporomandibular fluid.

A batch separation procedure has been developed for retrieval of tumor necrosis factor (TNF) alpha from the microliter volumes of fluid isolated from the human temporomandibular joint (TMJ). Paramagnetic beads coated with monoclonal antibodies for TNF were used. The beads, and bound TNF, were recovered from solution with the aid of a magnetic field. The amount of bead-bound TNF was quantified using an immuno-based assay developed in this laboratory called the cluster assay. The cluster assay was specific for TNF and linear up to 10 ng. Using these methods we found that TMJ fluid contained 0.2-4.2 ng per 100 microliters of fluid with a mean value of 1.9 ng and a standard deviation of 1.1 ng. This study demonstrates the utility of batch immunomagnetic separation for the concentration and purification of proteins, and the cluster assay for quantification of proteins from microliter volumes of body fluids.

Antibodies, Monoclonal↗
PubMed1996

Immunomagnetic separation and PCR detection show Shigellae to be common faecal agents in children from urban marginal communities of Costa Rica.

Culture and immunomagnetic separation-polymerase chain reaction assays (IMS-PCR) were used to isolate and identify Shigella flexneri, S. dysenteriae type I and S. sonnei in faeces from 250 children up to 5 years and from their mothers (n = 143) selected at random from a large urban marginal community of Costa Rica. Children hospitalized because of severe diarrhoea (n = 110) were also studied. Only S. flexneri, mainly serotype 2a, and S. sonnei were found by culture. All specimens in which Shigella was cultured were also positive by the corresponding IMS-PCR. S. flexneri was isolated by culture from 1 (0.7%) mother and 4 (1.6%) community children. S. sonnei was found in 2 (0.8%) children. An additional 12 S. flexneri and four S. sonnei in the community children were found by IMS-PCR. In total, Shigella was cultured from 0.7% of mothers and 2.4% of children. By the IMS-PCR 2% of mothers and 8% of children were positive. S. flexneri was isolated by culture from 14 (12.7%) hospitalized children and S. sonnei from 1 (0.9%). An additional 11 S. flexneri and three S. sonnei were found by IMS-PCR. In total, Shigella was cultured from 13.6% of hospitalized children. By the IMS-PCR 26% of them were Shigella positive. Thus IMS-PCR was more than twice as effective in diagnosing shigellae as culture. Twelve (60%) Shigella positive community children were above 3-years-old and 25% of them were under one year. Seven (35%) of the Shigella positive children had dysenteric and 9 (45%) normal stools. Half of the Shigella infected community children had been weaned before the 3 months of age. By the age of 5 months, 90% of them were already weaned. Seventeen (59%) of the hospitalized Shigella positive children were under 1 year of age. The stools were watery or semiliquid in 13 (45%) and dysenteric in 12 (41%) of them. We conclude that shigellosis is common in Costa Rica and represents an important cause of severe infant diarrhoea requiring hospitalization.

Adult↗
PubMed1998

Immunomagnetic separation and flow cytometry for rapid detection of Escherichia coli O157:H7.

A rapid method for detecting Escherichia coli O157:H7 combining immunomagnetic beads (IMB) and flow cytometry was developed. Labeling antigens separated by IMB with fluorescent antibody enabled the detection of < 10(3) CFU bacteria per ml in pure culture. The optimum concentration of magnetic beads for flow cytometry was lower (ca. 10(5) particles per ml) than that reported for conventional IMB assay (more than 6 x 10(6) to 8 x 10(6) particles per ml). Immunomagnetic separation and flow cytometry (IMFC) were evaluated for detecting E. coli O157:H7 in the presence of a competing microorganism and for detecting antibodies in potassium phosphate buffer. The total assay time from separating antigens with IMB to analyzing with flow cytometry was about 1 h. IMFC detected 10(3) to 10(4) CFU of E. coli O157:H7 per ml in ground beef enrichment broth and could effectively discriminate between E. coli O157:H7 and competing natural flora. The new assay system provides another approach to separation and detection of low populations of pathogens and shows potential for detecting low concentrations of toxins and other soluble antigens directly from food in a short time.

Animals↗
PubMed2000

PCR identification of Salmonella cells in food and stool samples after immunomagnetic separation.

The purpose of the present study was to investigate the application of various sample preparation methods (cell washing before lysis, purification of DNA using phenol extraction method, immunomagnetic separation-IMS) for the final PCR identification of Salmonella cells. The presence of PCR inhibitors in processed food products (milk powder and dried eggs) can be the cause of false-negative results in PCR without IMS of target cells. It was also demonstrated that IMS-PCR was successfully used for identification and quick confirmation of untypical Salmonella strains isolated from human stool samples and rabbit meat. However, IMS cannot eliminate intracellular PCR inhibitors present in immunoseparated Salmonella cells. These inhibitors must be taken into consideration in evaluation of PCR procedure.

Animals↗
PubMed2000

Evaluation of immunomagnetic separation for recovery of Cryptosporidium parvum and Giardia duodenalis from high-iron matrices.

In this study we examined the recovery of Cryptosporidium parvum and Giardia duodenalis from matrices containing various concentrations of dissolved iron. The organisms were recovered by using the immunomagnetic separation-immunofluorescent assay method, and the levels of recovery were compared to the dissolved iron concentrations. The levels of recovery of C. parvum decreased sharply at dissolved iron concentrations greater than 4 mg/liter, while the levels of recovery of G. duodenalis decreased sharply at concentrations greater than 40 mg/liter.

Animals↗
PubMed1990

A new method for isolation of reticulocytes: positive selection of human reticulocytes by immunomagnetic separation.

A method for isolating pure reticulocytes from leukocyte-depleted blood of normal persons is presented. The separation was achieved using an immunomagnetic technique. A monoclonal mouse antibody against human transferrin receptor was bound to magnetic beads conjugated with sheep antimouse antibody. The recovery of reticulocytes from peripheral blood was 15% to 42%. Blood used for isolation of reticulocytes could be stored for 4 days at 22 degrees C without altering the yield of reticulocytes. At 37 degrees C incubation, the reticulocytes matured rapidly and the transferrin receptor was found to have a half-life of 16 hours. The activity of several enzymes and the amount of creatine and hemoglobin A1C were measured both in the reticulocytes and peripheral blood. Of the enzymes, porphobilinogen deaminase had the best discriminatory power with a ratio of 8.8 between reticulocytes and peripheral red blood cells. The ratio for creatine was 16.7. The ability to isolate pure human reticulocytes, released after normal erythropoiesis, will offer new possibilities in the study of these cells.

Adult↗
PubMedDate not supplied

Immunomagnetic separation of adult human olfactory neural progenitors.

Olfactory neuroepithelium (ONe) has lifelong regenerative capacity owing to the presence of mitotically active progenitors. The accessibility of ONe makes it a unique source of progenitors for cell replacement strategies in the CNS. We have established lines of neurosphere forming cells (NSFCs) from adult postmortem ONe and patients undergoing nasal sinus surgery by endoscopic biopsy. These heterogeneous lines are composed primarily of an immature neuronally restricted and a small glial restricted subpopulation. More homogeneous subpopulations of the NSFCs are essential for detailed study of factors influencing their lineage restriction. Immunomagnetic bead separation using an antibody against tyrosine kinase (Trk) receptors (Trk-pan, which recognizes Trk-A, B, C) resulted in viable, enriched positive and negative subpopulations that could be analyzed immunocytochemically. The positive cells remained positive for the first week after which the number of Trk-pan expressing cells decreased. The negative subpopulation began to express Trk-pan immunoreactivity after five days in vitro. Both subpopulations reverted to the heterogeneous composition after two weeks. Furthermore, most NSFCs were positive for Trk-B, a few for Trk-A, while no reactivity was observed for Trk-C. Because NSFCs produce brain derived neurotrophic factor (BDNF) and express Trk B, the specific receptor for BDNF, it is likely that population dynamics are under a paracrine and/or autocrine regulatory mechanism. Lineage restriction analysis demonstrated that the isolated subpopulation had a restriction potential equivalent to the original heterogeneous population. These studies characterize further the NSFCs and support the future potential therapeutic use of ONe-derived progenitors for CNS injury and neurodegenerative disorders.

Adult↗