Immunomagnetic Separation Method Research Articles

The prevalence of verocytotoxin-producingEscherichia coli(VTEC) andE. coliO157:H7 in beef in Sweden determined by PCR assays and an immuno-magnetic separation (IMS) method

The number of cases of enterohaemorrhagicEscherichia coli(EHEC) in Sweden has increased dramatically since 1995, but no source of the infections has been identified. The prevalence of verocytotoxin-producingE. coli(VTEC), includingE. coliO157:H7 in imported and domestic raw beef in Sweden, was determined by PCR assays and immuno-magnetic separation (IMS, Dynal). VTEC was detected by a multiplex PCR directed at sequences on the vt1 and vt2 genes, and the prevalence ofE. coliO157:H7 was estimated by the IMS assay and a SZ-PCR which detectsE. coliO157:H7 (and some strains of O157:NM, O55:H7 and O55:NM). VTEC was detected in 15.055% (57/368) and 1.051% (6/543) of the imported and domestic beef samples, respectively, by the multiplex PCR. E. coliO157:H7 was detected in 0.053% and 2.054% of the imported beef samples by the IMS and SZ-PCR assays, respectively. In domestic beefE. coliO157:H7 was not detected by either method, indicating that the occurrence in Swedish beef was less than 0.056% with a 95% certainty. The overall prevalence of VTEC was estimated to be 4.050%, by adjusting for the relative proportion of imported and domestic beef in Sweden and, similarly, the overall prevalence ofE. coliO157:H7 was 0.0506% (IMS) or 0.055% (SZ-PCR). We propose that a useful strategy to analyse a large number of food samples for VTEC andE. coliO157:H7 is an initial screening of samples using the multiplex VT-PCR in conjunction with enrichment and buoyant density centrifugation, followed by further analyses on the fraction of VT-positive samples using either the IMS method or SZ-PCR.

Isolation ofMycobacterium paratuberculosisfrom Milk by Immunomagnetic Separation

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

Isolation of blood-borne epithelium-derived c-erbB-2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients.

Clinical studies including thousands of breast cancer patients have shown that c-erbB-2 is amplified and overexpressed in 20-30% of invasive human breast cancers and that it is associated with distant metastasis in specified patient subgroups. To isolate and characterize hematogeneously spreading c-erbB-2-positive epithelium-derived cells from the peripheral blood of breast cancer patients, a combined buoyant density gradient and immuno-magnetic separation method has been used. The method utilizes a biotinylated anti-cytokeratin monoclonal antibody (MAb) for capturing the epithelium-derived cells. The expression of c-erbB-2 by the captured cells was detected using an anti-c-erbB-2 rabbit antibody (21N) coupled to an anti-rabbit gold-labeled anti-body, whereby immunoenzymatic cytokeratin staining was performed using a silver-enhanced immunogold double staining protocol. In total, 29 of the 46 patients tested had either cytokeratin (24/29) or cytokeratin/c-erbB-2 (19/29) positive clustered cells in their peripheral blood. We thus report here the presence and the frequency of clone-specifically stained clustered cells in the peripheral blood of breast cancer patients. The frequency of cytokeratin/c-erbB2 double-positive clustered cells in the peripheral blood was on average 10 times higher than that of double-positive single cells. The numbers of cytokeratin/c-erbB-2 double-positive clustered cells were positively correlated with the stage of tumors. Results of in vitro motility experiments using single and clustered cells from primary breast cancer tissue strongly support the assumption that cytokeratin/c-erbB-2 double-positive clustered cells have a high potential for locomotion. We suggest that blood-borne epithelium-derived c-erbB-2-positive clustered cells are the possible precursor cells responsible for the formation of distant metastases and bone marrow micrometastases.

Immunomagnetic isolation of human developing motor neurons.

Human motor neuron (MN) isolation provides a critical tool to study neurophysiological properties and the effects of molecules of clinical relevance on isolated neurons. We developed an immunomagnetic separation technique based on specific MN antigen recognition for nerve growth factor receptor (p75-NGFR). We cultured an average of 250,000 cells from the anterior horns of a single cord (four specimens at postconception Weeks 6.0, 7.2, 8.0, and 8.3). At day 7 in vitro (DIV), choline acetyltransferase (ChAT) and/or p75-NGFR-expressing cells (MNs) represented 72 +/- 2% of the total growing cells. MNs survived for at least 4 weeks in biochemically defined medium. The immunomagnetic separation method has been demonstrated to be effective, reproducible, and quantitative for separation of MNs.

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.

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

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

Detection of Escherichia cole O157 in French food samples using an immunomagnetic separation method and the VIDAS E. coli O157.

Two commercially available screening methods, an automated enzyme-linked fluorescent immunoassay (VIDAS E. coli O157) and an immunomagnetic separation followed by culture onto cefixime tellurite sorbitol MacConkey agar (CT-SMAC), were compared for detection of Escherichia coli O157 in naturally and artificially contaminated food samples. A total of 250 naturally contaminated food samples, including raw milk cheeses, poultry, raw sausages and ground beef retail samples, were examined. Four poultry, one raw sausage and one ground beef sample were found to be positive for E. coli O157 by both methods. Of the six positive samples, five were shown to contain sorbitol-positive, O157-positive, H7-negative, motile and non-verotoxin-producing E. coli.

Immunomagnetic separation of Cryptosporidium oocysts from water samples: round robin comparison of techniques

A prototype immunomagnetic separation (IMS) technique was tested in five laboratories, which undertake routine analysis of water samples for Cryptosporidium oocysts, by comparing the recovery efficiency of the IMS technique with techniques in current use (the “Blue Book” Standing Committee of Analysts, SCA, method and flow cytometry, FCM). In very low turbidity samples (clean waters) of both 1ml and 10ml volumes the IMS demonstrated significantly better results than both SCA and FCM methods. Not only were higher oocyst recovery efficiencies reported but variation in recovery efficiency was reduced and fewer negative results were reported from oocyst-seeded samples than with the other two techniques. In trials with clean water or low turbidity water, FCM was the technique which most consistently reported negative results in oocyst-seeded samples and for clean water this difference was found to be statistically significant. When the water sample was turbid the recovery efficiency of the IMS technique diminished. The results suggest that the IMS technique is affected to different extents by different material constituents in water concentrates and that FCM is apparently less affected by interfering particulate matter. Despite the potential difficulties with the IMS method with turbid water samples, the results from these trials indicate that this technique would be a very useful addition to the armoury of methods for the concentration of oocysts from water samples and was considered by the trial participants to be simple, user-friendly and applicable to the processing of multiple samples simultaneously.

Immunomagnetic separation for enhanced flagellar antigen phase inversion in salmonella.

Immunomagnetic separation (IMS) was used to reduce delays in serotyping caused by slow flagellar phase inversion of diphasic salmonellas. Some 375 strains of 11 salmonella serotypes were examined using IMS. The mean time for successful flagellar phase inversion was reduced from 4.3 (range 1-18) d to 1.0 (range 1-3) d using the IMS method, and in a further experiment phase inversion was achieved within 8 h in 112 of a further 117 strains representing four salmonella serotypes.

Immunomagnetic separation of Cryptosporidium oocysts from water samples: round robin comparison of techniques

A prototype immunomagnetic separation (IMS) technique was tested in five laboratories, which undertake routine analysis of water samples for Cryptosporidium oocysts, by comparing the recovery efficiency of the IMS technique with techniques in current use (the “Blue Book” Standing Committee of Analysts, SCA, method and flow cytometry, FCM). In very low turbidity samples (clean waters) of both 1ml and 10ml volumes the IMS demonstrated significantly better results than both SCA and FCM methods. Not only were higher oocyst recovery efficiencies reported but variation in recovery efficiency was reduced and fewer negative results were reported from oocyst-seeded samples than with the other two techniques. In trials with clean water or low turbidity water. FCM was the technique which moat consistently reported negative results in oocyst-seeded samples and for clean water this difference was found to be statistically significant. When the water sample was turbid the recovery efficiency of the IMS technique diminished. The results suggest that the IMS technique is affected to different extents by different material constituents in water concentrates and that FCM is apparently less affected by interfering particulate matter. Despite the potential difficulties with the IMS method with turbid water samples, the results from these trials indicate that this technique would be a very useful addition to the amsoury of methods for the concentration of oocysts from water samples and was considered by the trial participants to be simple, user-friendly and applicable to the processing of multiple samples simultaneously.

Evaluation of Methods for the Isolation and Detection of Escherichia coli O157:H7 from Minced Beef

This study has evaluated enrichment and detection procedures for the isolation and detection of Escherichia coli O157:H7 inoculated into minced beef, i. e., 24hr enrichment in modified EC broth containing novobiocin (mEC+n) followed by plating onto sorbitol IPA bile salt agar (SIB), sorbitol MacConkey agar (SMAC), cefixime-tellurite SMAC (CT-SMAC) and Rainbow O157 agar, or immunomagnetic separation (IMS) followed by plating onto these four kind of media. The recovery of E. coli O157:H7 was greater on CT-SMAC than SMAC, SIB or Rainbow O157 Agar. The protocol using mEC+n incubated at 42°C, followed by IMS and inoculation onto CT-SMAC was the most succesful procedure. However, 24hr enrichment in mEC+n followed by CT-SMAC was almost as effective as the IMS method for the isolation of E. coli O157:H7.

Immunomagnetic-electrochemiluminescent detection of Escherichia coli O157 and Salmonella typhimurium in foods and environmental water samples

Hemorrhagic Escherichia coli O157:H7 strains and other virulent enteric pathogens can pose a serious health threat in tainted meats, poultry, and even drinking water. Traditional culture-based methods for assay of enteric pathogens in foods and water sources are relatively slow, and results can be ambiguous. Immunomagnetic separation (IMS) and detection methods have been investigated and appear promising for rapid bacterial assay of foods and environmental samples. In this work, a commercial sensor which combines IMS with electrochemiluminescence (ECL) detection is evaluated for detection of E. coli O157 and Salmonella typhimurium in foods and fomites. Results indicate that detection limits are in the range of 100 to 1,000 bacteria per ml in pristine buffer for E. coli O157 and S. typhimurium, respectively, or 1,000 to 2,000 bacteria per ml in food samples (depending on the sample) and that total processing and assay time is rapid (< 1 h) even in food samples. An immunologic "hook" or high-antigen-concentration prozone effect was observed above 10(4) and 10(5) bacteria per ml for E. coli O157 and S. typhimurium, respectively. IMS was accomplished in milk, juices, serum, supernatant fluids from ground beef, finely minced chicken, and fish suspensions as well as several freshwater sources and followed by ECL assay. Some samples, especially fish, gave unexpectedly high background ECL. Conversely, low ECL intensity was observed in nonfat and 2% fat milk samples, which appeared to be related to binding or entrapment of the antibody-coated magnetic beads by particulates in the milk, as revealed by microscopy. Results of this evaluation suggest the feasibility of immunomagnetic-ECL methodology for rapid, sensitive, and facile preliminary screening of various foods and fomites for the presence of virulent enteric pathogens.

Sensitive detection of group A rotaviruses by immunomagnetic separation and reverse transcription-polymerase chain reaction

An immunomagnetic separation (IMS) method was developed for concentrating rotaviruses from environmental samples, as well as a reverse transcription-polymerase chain reaction (RT-PCR) for the sensitive and specific detection of group A rotaviruses. Magnetic beads were coated with monoclonal antibodies directed against the group-specific, inner capsid protein (VP6) and subsequently used to capture and purify the virus with the help of a magnet. The genome was made available for RT by heat-disrupting the viral particles. A single 40-cycle PCR was as sensitive as a nested PCR, both detecting 0.005 PFU of the Wa strain, corresponding to approximately 5 particles as indicated by EM. The nested PCR was positive for all the group A strains tested, but negative for group C rotaviruses and other RNA viruses. The IMS-RT-PCR method functioned satisfactorily with virus seeded out in fresh water samples; with sea water, the IMS removed most, but not all, inhibiting activity.

Expression of adhesion molecules on human hematopoietic progenitor cells at different maturational stages.

In this report we examined the expression of several adhesion molecules on human hematopoietic progenitor cells at different maturational stages. Human hematopoietic progenitor cell-enriched fractions were prepared from bone marrow cells by depleting lymphocytes and monocytes (CD2+, CD14+ and CD19+ cells). These cells were separated into adhesion molecule-positive and -negative cell populations by immunomagnetic separation methods and then assessed for their ability to form various colony forming cells (CFC). CD44 and CD49d were expressed on multipotent hematopoietic progenitor cells, or mixed colony forming units (CFU-Mix), erythroid burst forming units (BFU-E), granulocyte-macrophage CFU (CFU-GM) and erythroid CFU (CFU-E). Leu8 was expressed on CFU-Mix, BFU-E and some populations of CFU-GM, but not CFU-E. CD11a was expressed on some populations of CFU-Mix, CFU-GM and BFU-E. CD54 was expressed only on some populations of CFU-GM. These results suggest that Leu8, CD44, CD49d and CD11a appear to play important roles in the differentiation and proliferation of human hematopoietic progenitor cells at different maturational stages in the bone marrow microenvironment.

Rapid isolation of Escherichia coli O157:H7 from enrichment cultures of foods using an immunomagnetic separation method

A method employing immunomagnetic beads was developed for the isolation of Escherichia coli O157:H7. Magnetic beads precoated with sheep anti-rabbit IgG were coated with rabbit antiserum against E. coli O157. The level of bacteria, amount of beads, amount of antibody coating the beads and incubation time were varied to determine their effects on recovery of the organism. The sensitivity of the test for recovery of E. coli O157:H7 was 1 × 101 cfu ml−1. The organism was selectively recovered from 24 h enrichment cultures of meat products in which the initial inoculum level was 1 bacterium g−1. Sorbitol negative colonies were selected from Sorbitol Mac-Conkey agar and were positively identified as E. coli using a direct fluorescent antibody procedure. The results indicate that immunomagnetic separation is a convenient and rapid method for the isolation of E. coli O157:H7 from foods.

Identification of mRNA for PDGF B‐chain in human megakaryocytes isolated using a novel immunomagnetic separation method

A rapid and simple method has been developed for the separation of pure populations of intact human megakaryocytes from whole bone marrow. Megakaryocytes were specifically recognized using monoclonal antibodies coupled to magnetizable articles. Cells labelled with the magnetizable probe were separated from unlabelled cells by introduction of a magnetic field. The technique yields megakaryocyte suspensions with a purity of greater than 98%. Electron microscope examination showed that the ultrastructure of the isolated megakaryocytes was well preserved. Using this method of cell purification, we have investigated expression of the gene for platelet-derived growth factor (PDGF). RNA was isolated from cells harvested from either ribs or posterior iliac crest. The RNA was spotted onto nitrocellulose, and then hybridized using a c-sis riboprobe specific for PDGF B chain mRNA. We demonstrate that mRNA for PDGF B-chain is identifiable in samples of 50,000 cells. We conclude that PDGF is synthesized by the megakaryocyte.