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Results for “Immunomagnetic Separation”

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PubMed2001

A novel method for isolation of neutrophils from murine blood using negative immunomagnetic separation.

Inappropriate neutrophil activation has been implicated in the pathology of several clinically important inflammatory conditions. Although murine models are extensively used in the investigation of such pathological processes, a reliable method by which viable, quiescent neutrophils can be isolated from murine blood has not been developed. Here we describe a novel method based on negative immunomagnetic separation, which yields highly pure populations of murine neutrophils. Blood is incubated with a cocktail of antibodies against specific cell markers on unwanted cells, and then with secondary antibody-coated magnetic beads. After running the preparation through a column within a magnetic field, labeled cells are retained, and a neutrophil-rich effluent is collected. This method yields a >95% pure suspension of >97% viable neutrophils, recovering approximately 70% of neutrophils from whole blood. Flow cytometric analysis shows little difference in surface L-selectin and CD18 expression on isolated neutrophils compared with neutrophils in whole blood, indicating that neutrophils are minimally activated bythe isolation process. Stimulation with phorbol 12-myristate 13-acetate (PMA) reduced L-selectin andincreased CD18 expression. Isolated neutrophilsmigrate under agarose in response to fMLP, and fluorescently labeled neutrophils transfused into recipient mice interact with postcapillary venules in a manner comparable to endogenous leukocytes. These findings show that neutrophils isolated using this method can be used for inflammatory studies in vitro and in vivo.

Animals↗

PubMed2001

Comparison of culture, PCR and immunoassays for detecting Escherichia coli O157 following enrichment culture and immunomagnetic separation performed on naturally contaminated raw meat products.

The aims of this study were (i) to evaluate the specificity and sensitivity of three previously described PCR assays for the detection of Escherichia coli O157 and, (ii) to compare PCR, culture, and two visual immunoassays (VIAs), BioSign and Path-Stik, for detecting E. coli O157 after enrichment culture and immunomagnetic separation (IMS) performed on various naturally contaminated raw beef, lamb and mixed meat products. Twelve sorbitol nonfermenting (SNF) verocytotoxin-producing (VT +) E. coli O157, 6 SNF VT - E. coli O157, 4 sorbitol fermenting (SF) VT + E. coli O157, 3 SF VT - E. coli O157, 23 E. coli belonging to 17 other serogroups and 12 organisms of other species were used to check the specificity of PCR reactions. Only one primer pair generated amplimers only with E. coli O157 and was used for all subsequent work. After enrichment culture and on inoculated minced beef samples, PCR was as sensitive as culture for detecting 9 of the 12 strains of E. coli O157, but up to 4 log10 more sensitive than culture for detecting three strains. Of the 120 samples of naturally contaminated meat products examined, 80 (67%) were positive by PCR, 70 (58%) were positive by BioSign, 69 (58%) were positive by culture and 67 (56%) were positive by Path-Stik. Although both VIAs lacked sensitivity when compared to PCR, both compared favourably with culture and both were extremely rapid and easy to perform, giving a result in less than 15 min. Eleven samples were positive by PCR and both VIAs, but negative by culture because culture plates were heavily overgrown with SF organisms, making detection of any E. coli O157 present impossible.

Animals↗

PubMed2001

The detection of the HLA-B27 antigen by immunomagnetic separation and enzyme-linked immunosorbent assay-comparison with a flow cytometric procedure.

The HLA-B27 antigen is an important genetic marker in ankylosing spondylitis (AS). Methods for the detection of B27 include the microlymphocytotoxicity test and, more recently, flowcytometry (FC). Here, we describe a new method, IMS-ELISA, for measuring the B27-antigen. It combines immunomagnetic separation (IMS), to obtain B27-positive cells from whole blood samples, with an enzyme-linked immunosorbent assay (ELISA) as a read-out. IMS-ELISA was tested on 367 samples obtained from five different hospitals in Taiwan. The sensitivity, specificity and accuracy of the method were compared with FC. Any conflicting data between IMS-ELISA and FC was confirmed by HLA-DNA typing via PCR-SSP (polymerase chain reaction-sequence specific primers). Overall, the results for sensitivity, specificity and accuracy obtained by IMS-ELISA and FC did not show any significant difference (p>0.05). However, when considering laboratory time, cost, ease of operation and the screening of large samples for HLA-B27, the IMS-ELISA was superior to the FC method. We conclude that IMS-ELISA may be used as a fast screening method for HLA B27 detection.

Enzyme-Linked Immunosorbent Assay↗

PubMed2004

Detection of bone marrow micrometastasis in esophageal cancer patients by immunomagnetic separation.

Detection of micrometastasis to the bone marrow can predict widespread disease and a poor prognosis of cancer patients. The purpose of this study was to evaluate the clinical significance of detecting micrometastasis in the bone marrow of esophageal cancer patients. Bone marrow and peripheral blood samples were obtained from 52 squamous esophageal cancer patients at the time of surgery. These samples were enriched by immunomagnetic separation and immunostained with an anti-cytokeratin antibody. Expression of vascular endothelial growth factor (VEGF) and cyclin D1 was examined in the primary tumors. Cytokeratin-positive cancer cells were observed in the bone marrow of 13 (25%) out of 52 patients. Among them, three patients also had cancer cells in the peripheral blood. The presence of bone marrow micrometastasis was correlated with lymph node metastasis (pN) but not associated with depth of tumors (pT). Detection of bone marrow micrometastasis was significantly correlated with VEGF expression of the primary tumors. Cumulative survival of patients with bone marrow micrometastasis was significantly lower than that of patients without it (p=0.0008). Hematogenous recurrence was more frequent in patients with bone marrow micrometastasis than in those without it (p=0.0004). Three patients who had local or dissemination recurrence did not have bone marrow micrometastasis. Detection of cancer cells in the bone marrow might be an indicator of early hematogenous metastasis in esophageal cancer patients. Intensive postoperative chemotherapy seems to be indicated for these patients.

Aged↗

PubMed2003

Detection of bone marrow micrometastasis in gastric cancer patients by immunomagnetic separation.

BACKGROUND: Micrometastasis to the bone marrow can predict widespread disease and a poor prognosis of cancer patients after surgery. The purpose of this study was to evaluate the clinical significance of detecting micrometastasis in the bone marrow of gastric cancer patients. METHODS: Bone marrow and peripheral blood samples were obtained from 53 gastric cancer patients at the time of surgery. These samples were enriched by immunomagnetic separation and immunostained with an anti-cytokeratin antibody. Expression of vascular endothelial growth factor and erbB-2/HER2 was examined in the primary tumors. RESULTS: Cytokeratin-positive cancer cells were observed in the bone marrow of 16 (30%) of 53 patients. Among them, two patients also had cancer cells in the peripheral blood. The presence of bone marrow micrometastasis was correlated with the depth of invasion and lymph node metastasis but was not associated with peritoneal dissemination. Detection of bone marrow micrometastasis was not correlated with vascular endothelial growth factor or HER2 expression in the primary tumors. Four patients with micrometastasis had recurrence in the liver or lungs, but this did not occur in patients without micrometastasis. CONCLUSIONS: Detection of cancer cells in the bone marrow might be an indicator of postoperative hematogenous metastasis in gastric cancer patients.

Adult↗

PubMed2006

Sensitivity of an immunomagnetic-separation-based test for detecting Escherichia coli O26 in bovine feces.

The sensitivity of a test for cattle shedding Escherichia coli serogroup O26 was estimated using several fecal pats artificially inoculated at a range of concentrations with different E. coli O26 strains. The test involves the enrichment of fecal microflora in buffered peptone water, the selective concentration of E. coli O26 using antibody-coated immunomagnetic-separation beads, the identification of E. coli colonies on Chromocult tryptone bile X-glucuronide agar, and confirmation of the serogroup with E. coli serogroup O26-specific antisera using slide agglutination. The effective dose of E. coli O26 for an 80% test sensitivity (ED(80)) was 1.0 x 10(4) CFU g(-1) feces (95% confidence interval, 4.7 x 10(3) to 2.4 x 10(4)). Differences in test sensitivity between different E. coli O26 strains and fecal pats were also observed. Individual estimates of ED(80) for each strain and fecal pat combination ranged from 4.2 x 10(2) to 4.8 x 10(5) CFU g(-1). These results suggest that the test is useful for identifying individuals shedding a large number of E. coli O26 organisms or, if an appropriate number of individuals in a herd are sampled, for identifying affected herds. The study also provides a benchmark estimate of sensitivity that can be used to compare alternative tests for E. coli O26 and a methodological approach that can be applied to tests for other pathogenic members of the Enterobacteriaceae and other sample types.

Animals↗

PubMed2003

Immunomagnetic separation and detection of Salmonella cells using newly designed carriers.

Magnetic nonporous poly(HEMA-co-EDMA) and poly(HEMA-co-GMA) microspheres were prepared by dispersion copolymerisation of 2-hydroxyethyl methacrylate (HEMA) and ethylene dimethacrylate (EDMA) or glycidyl methacrylate (GMA) in the presence of magnetite. They were functionalized by polyclonal Salmonella antibodies via the trichlorotriazine method. Salmonella cells were then successfully identified using cultural and polymerase chain reaction (PCR) methods after their immunomagnetic separation. The PCR sensitivity of target cell detection was negatively influenced by the presence of some compounds used in the process of particle preparation. In some cases, magnetic poly(HEMA-co-EDMA) microspheres with immobilized proteinase K were used for degradation of intracellular inhibitors present in Salmonella cells.

Electrophoresis, Agar Gel↗

PubMedDate not supplied

Immunocytochemical detection of circulating esophageal carcinoma cells by immunomagnetic separation.

BACKGROUND: Recent developments in detection of micrometastasis have revealed a considerable incidence of systemic disease in patients who would previously have been diagnosed as having solid tumours only. The purpose of this study was to investigate the prevalence and clinical significance of circulating carcinoma cells in patients with esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: Peripheral blood samples from 47 primary ESCC patients were enriched by immunomagnetic separation (IMS) using Ber-EP4-conjugated beads (Dynabeads anti-epithelial cell) and immunostained with an anti-cytokeratin (anti-CK) antibody. We assayed samples from 12 patients to compare the detection of CK-reactive cells using IMS with the detection of CK 19 mRNA using a reverse transcriptase-polymerase chain reaction (RT-PCR) method. RESULTS: CK-reactive cells were observed in 18 out of 47 patients (38%). The detection rate was closely correlated with the stage of disease (TNM stage) (p = 0.0017). In 33 patients who underwent esophagectomy, 4 out of 7 patients (57%) positive for CK cells and only 2 out of 26 patients (7.7%) negative suffered from recurrence in the distant lymph nodes or lungs (p = 0.0108). When IMS and RT-PCR were compared, all 3 samples that were CK-positive by IMS had CK 19 mRNA detected by RT-PCR. However, the other 4 samples with CK 19 mRNA by RT-PCR were negative for CK cells by IMS. Of 7 patients positive for CK-reactive cells before chemotherapy or chemoradiotherapy, 4 patients negative after treatment survived but 3 patients who remained positive died within 6 months. CONCLUSION: Immunocytochemical detection of circulating carcinoma cells by IMS may be a specific method for the diagnosis of systemic disease and for monitoring treatment response.

Adult↗

PubMed2000

Escherichia coli O26 detection from foods using an enrichment procedure and an immunomagnetic separation method.

We found effective enrichment procedures for detecting Escherichia coli O26 in foods using methods that are used for E. coli O157. Ground beef or radish sprouts inoculated with approximately 6 colony-forming units of E. coli O26 were homogenized in 225 ml of various broths. After static incubation at 37 degrees C or 42 degrees C for 6 h or 18 h, we isolated the inoculated bacterium by plating onto Rainbow Agar O157 with novobiocin. In combination with the immunomagnetic separation method, E. coli O26 was isolated from all samples by using enrichment in tryptone soy broth at 37 degrees C for 6 h and in modified E. coli broth with novobiocin (mEC + n) at 42 degrees C for 18 h in ground beef and radish sprouts, respectively. Enrichment in mEC + n at 42 degrees C for 18 h was effective for isolating both E. coli O26 and E. coli O157 from both ground beef and radish sprouts.

Animals↗

PubMed2001

A comparison of immunomagnetic separation and culture, Reveal and VIP for the detection of E. coli O157 in enrichment cultures of naturally-contaminated raw beef, lamb and mixed meat products.

AIMS: The aims of this study were (i) to evaluate the specificity and sensitivity of three previously described PCR assays for the detection of E. coli O157 and, (ii) to compare PCR, culture, and two visual immunoassays (VIAs), BioSign and Path-Stik, for detecting E. coli O157 after enrichment culture and immunomagnetic separation (IMS) performed on various naturally contaminated raw beef, lamb and mixed meat products. METHODS AND RESULTS: Twelve sorbitol non fermenting (SNF) verocytotoxin-producing (VT+) E. coli O157, 6 SNF VT- E. coli O157, 4 sorbitol fermenting (SF) VT+ E. coli O157, 3 SF VT- E. coli O157, 23 E. coli belonging to 17 other serogroups and 12 organisms of other species were used to check the specificity of PCR reactions. Only one primer pair generated amplimers only with E. coli O157 and was used for all subsequent work. After enrichment culture and on inoculated minced beef samples, PCR was as sensitive as culture for detecting 9 of the 12 strains of E. coli O157, but up to 4 log10 more sensitive than culture for detecting three strains. Of the 120 samples of naturally contaminated meat products examined, 80 (67%) were positive by PCR, 70 (58%) were positive by BioSign, 69 (58%) were positive by culture and 67 (56%) were positive by Path-Stik. Eleven samples were positive by PCR and both VIAs, but negative by culture because culture plates were heavily overgrown with SF organisms making detection of any E. coli O157 present impossible. CONCLUSIONS: PCR and both VIAs compared well with culture of beads to CT-SMAC for detecting E. coli O157 after enrichment culture and IMS. PCR appeared to be the most sensitive method, but needed specialised equipment and was also the most expensive, laborious and technically demanding technique. Although lacking the sensitivity of PCR, the VIAs were of comparable sensitivity to culture and were extremely quick and easy to perform giving a result in less than 15 minutes. SIGNIFICANCE AND IMPACT OF THE STUDY: Culture techniques may fail to detect E. coli O157 retrieved by IMS due to overgrowth with other organisms.

Cell Culture Techniques↗

PubMed2000

The detection of Salmonella using a combined immunomagnetic separation and ELISA end-detection procedure.

The aim of this study was to develop a rapid immunoassay to detect Salmonella bacteria. Skimmed milk powder (SMP) in buffered peptone water was inoculated with six Salmonella strains (Salm. typhimurium, Salm. virchow, Salm. enteritidis, Salm. give, Salm. ealing and Salm. arizonae) at three inoculum levels (about 2-200 cfu 25 g(-1) SMP) and incubated (37 degrees C) overnight. Heat-treated salmonella cells were immobilized on paramagnetic particles and detected within 3 h using the Salmonella genus-specific monoclonal antibody M105 in a microtitre plate based assay. The rapid Salmonella detection method combining immunomagnetic separation and ELISA had a total isolation and detection time of less than 24 h, which is significantly shorter than the conventional techniques requiring 72-96 h. The technique had a sensitivity limit of 10(5)-10(6) cfu ml(-1).

Animals↗

PubMed1994

Use of ferrous sulphate and immunomagnetic separation to recover Salmonella enteritidis from raw eggs.

Contaminated eggs or foods containing eggs have been a source of food borne salmonellosis, with a significant proportion of these outbreaks being attributed to Salmonella enteritidis. Since the level of contamination in individual eggs or a pool of such eggs may be low, enrichment to increase cell numbers can take several days. Pre-enrichment of raw blended eggs which have been supplemented with ferrous sulphate at a concentration of 35 mg/l, for 6 h at 37 degrees C, significantly enhanced the growth of Salmonella. Using Dynabeads Anti-Salmonella (Dynal, Oslo), a new commercial product for selective enrichment of Salmonella from food samples, we have defined a protocol based on immunomagnetic separation, specially for raw eggs. The application of this protocol enables definitive detection of Salmonella enteritidis from eggs within 30 h.

Animals↗

PubMed2003

Analysis-only detection of Giardia by combining immunomagnetic separation and two-color flow cytometry.

BACKGROUND: Giardia is a protozoan parasite of concern to water utilities. Giardia detection relies on cyst isolation and confirmation with the use of fluorescence microscopy. It is of interest to develop a flow cytometric (FCM) method that reliably detects one cyst in 10 L of water. To date all available antibodies have targeted the same epitope on the cyst wall. To achieve a reliable method, two independent probes are required. METHODS: Giardia cysts were spiked into a backwash water sample with and without prior hybridization to peptide nucleic acid (PNA) probes. Immunomagnetic separation (IMS) as a pre-enrichment step was compared with filtration of the water sample. Cysts were recovered with two-color FCM. Those cysts hybridized with PNA and fluorescein isothiocyanate (FITC) were dual stained with monoclonal antibody (mAb) conjugated to phycoerythrin (PE); those not hybridized to PNA were dual stained with mAb-FITC and mAb-PE. RESULTS: A fourfold increase in fluorescent signal intensity was obtained when combining the mAb-PE and PNA probe compared with two-color antibody staining. When combined with IMS, Giardia was successfully identified by FCM, with no false positives detected. CONCLUSIONS: Analysis-only FCM detection of Giardia in water is feasible. Further method development incorporating PNA probe hybridization after IMS is necessary.

Animals↗

PubMed1996

Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods, and direct culture.

We examined 30 children with classical hemolytic-uremic syndrome (HUS) for the presence of enterohemorrhagic Escherichia coli (EHEC) strains in stool samples and determined the specific immune response to O157 lipopolysaccharide in acute-phase serum samples from these patients. EHEC O157 strains were isolated from stool samples of 18 (60%) of the patients, and non-O157 EHEC strains were isolated from 5 (17%) of the patients. For O157 strain isolation from stools, we introduced a selective enrichment step using O157-specific antibodies attached to paramagnetic particles (immunomagnetic separation [IMS] method). This procedure allowed the detection of O157 strains at 10(2) CFU/g of stool in the presence of 10(7) coliform background flora organisms. By using IMS followed by plating on sorbitol MacConkey (SMAC) agar and cefixime-tellurite SMAC (CT-SMAC) agar, O157 strains were detected in 18 samples, whereas colony hybridization detected a subset of 12 positive samples and direct culture on CT-SMAC or SMAC agar detected only 7. Three of the 18 O157-positive stools were negative by cytotoxicity assay performed with stool filtrates and by direct PCR with DNA extracted from stools. The IMS technique allowed the isolation of O157 strains from 18 of 20 patients with serological evidence for O157 infection. Apart from the increase in sensitivity in O157 detection compared with that of direct culture, the IMS technique also has the advantage of being less labor-intensive and less time-consuming than the molecular methods. IMS can therefore be considered an efficient method for wide-spread use in the detection of O157 strains in clinical microbiology laboratories. However, because a significant number of HUS cases were attributable to non-O157 EHEC serogroups, the use of additional methods besides IMS in the bacteriological diagnosis of HUS is necessary.

Antibodies, Bacterial↗

PubMed1997

A 5-h screening and 24-h confirmation procedure for detecting Escherichia coli O157:H7 in beef using direct epifluorescent microscopy and immunomagnetic separation.

An antibody-direct epifluorescent filter technique (Ab-DEFT) detected 100% of the raw ground beef samples inoculated with Escherichia coli O157:H7 cells (0.15 cells g-1) and incubated in a prewarmed, modified buffered peptone water (mBPW) non-selective enrichment broth for 5 h at 42 degrees C in an orbital shaking water bath (200 rev min-1). Over 50% of the microscopic fields viewed were positive (1-10 fluorescent cells field-1) in the Ab-DEFT. All positive screening results were confirmed within 24 h by subjecting 1 ml of the mBPW to the Dynabeads anti-E. coli O157 immunomagnetic separation procedure, followed by plating on MacConkey sorbitol agar containing 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. At this cell concentration, 41.7% of the inoculated samples were detected by the conventional method involving a 24-h selective enrichment. Exposure to viable cells before filtration was minimized by using a 0.58% formaldehyde concentration for 5 min at 50 degrees C (killed > 4.00 logs of E. coli O157:H7 cells) without affecting cell fluorescence.

Animals↗

PubMedDate not supplied

Detection of E. coli in beach water within 1 hour using immunomagnetic separation and ATP bioluminescence.

The contamination of beach waters occurs from the discharge of storm water and sanitary sewer overflows containing faecal material. Additional faecal material derives from discharge of animals and waterfowl. In order to protect public from exposure to faecal-contaminated water, it is required to test enteric indicators in beach water. The problem is that the traditional culture-based methods cannot meet this goal because it takes too long (>24 h), so the results are not available until a day later. A rapid method for testing beach water for Escherichia coli within 1 h has been developed. Immunomagnetic separation (IMS) and ATP bioluminescence were used for selective capture and quantification, respectively. This rapid method was compared to the current method (m-TEC) using beach water samples. The beach samples were prefiltered with a 20 microm pore size filter in order to remove algae, plant debris and large particles. The results showed that the prefiltration step did not trap the bacteria which were present in the original water samples. The prefiltered water was then passed through a 0.45 microm pore size filter for concentration. The deposited bacteria were resuspended and then mixed with superparamagnetic polystyrene beads (diameter of 0.6 microm) that were coated with E. coli antibodies. After IMS, the quantification of the E. coli was done by ATP bioluminescence. The results obtained with IMS-ATP bioluminescence correlated well with the plate count method (Rsq = 0.93). The detection limit of the assay was about 20 CFU/100 mL, which is well below the US EPA limits for recreational water. The entire procedure can be completed in less than 1 hour. The necessary equipment is portable and was tested on-site.

Adenosine Triphosphate↗

PubMed2001

Development and optimization of a novel immunomagnetic separation- bacteriophage assay for detection of Salmonella enterica serovar enteritidis in broth.

Salmonella is the second-leading cause of food-borne illness in most developed countries, causing diarrhea, cramps, vomiting, and often fever. Many rapid methods are available for detection of Salmonella in foods, but these methods are often insensitive or expensive or require a high degree of technical ability to perform. In this paper we describe development and characterization of a novel assay that utilizes the normal infection cycle of bacteriophage SJ2 for detection of Salmonella enterica serovar Enteritidis in broth. The assay consists of four main stages: (i) capture and concentration of target cells by using immunomagnetic separation (IMS); (ii) infection of the target bacterium with phage; (iii) amplification and recovery of progeny phage; and (iv) assay of progeny phage on the basis of their effect on a healthy population of host cells (signal-amplifying cells). The end point of the assay can be determined by using either fluorescence or optical density measurements. The detection limit of the assay in broth is less than 10(4) CFU/ml, and the assay can be performed in 4 to 5 h. The results of this study demonstrate that the IMS-bacteriophage assay is a rapid, simple, and sensitive technique for detection of Salmonella serovar Enteritidis in broth cultures which can be applied to preenriched food samples.

Bacteriological Techniques↗

PubMed1996

Detection of enterohaemorrhagic Escherichia coli O157:H7 in minced beef using immunomagnetic separation.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 has been recently recognized as a human pathogen associated with haemorrhagic colitis and haemolytic uraemic syndrome. Most outbreaks of haemorrhagic colitis resulted from the consumption of undercooked minced beef or raw milk. Dairy cattle have been identified as a reservoir of EHEC O157: H7. In this study E. coli O157 specific antibody, coated on magnetic beads, was used to concentrate and release EHEC O157:H7 from meat samples. A survey of retail fresh minced beef and hamburger samples using this procedure revealed that 3 (5%) of 58 beef samples were positive for EHEC O157:H7. Two of the strains produced both VT1 and VT2 verotoxins, and one produced only VT2. Immunomagnetic separation is a sensitive and simple technique for the isolation of E. coli O157 from food, and could be useful for a further elucidation of the epidemiology of this organism. The relatively high prevalence of EHEC O157:H7 in beef samples may constitute a risk for public health. Thus, a suitable epidemiologic control and effective methods of prevention should be applied.

Animals↗