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At least 19 recordsLinked to original sources
PubMed1993

Comparison of immunomagnetic beads coated with protein A, protein G, or goat anti-mouse immunoglobulins. Applications in enzyme immunoassays and immunomagnetic separations.

Immunomagnetic beads were prepared using either protein A (PA) or protein G (PG) coupled to magnetic beads for binding antibodies at their Fc region. The performance of these beads was compared with commercially available beads coated with goat anti-mouse (G alpha M) immunoglobulins. Both the PA- and PG-beads possessed a higher binding capacity than the G alpha M-beads for the monoclonal antibodies tested, although, PA bound weakly with some IgG1 antibodies. PA-beads were compared with G alpha M-beads in a magnetic enzyme immunoassay for the detection of mouse immunoglobulins as an alternative to a conventional capture ELISA. The magnetic enzyme immunoassay was characterized by a detection time of less than 60 min and a linear assay range from 5-10 to 500 ng/ml for G alpha M-beads and 5-10 to 1000 ng/ml for PA-beads. The capture ELISA was linear from 10 to 250 ng/ml. For immunomagnetic separation of Salmonella with immunomagnetic beads, PA-beads were superior to both PG- and G alpha M-beads. For specific isolation of bacteria from heterogeneous suspensions by immunomagnetic separation, PA- and PG-beads are preferable since G alpha M-beads crossreact with bacteria possessing proteins with Fc-binding activity.

Animals↗
PubMed1990

Detection of Listeria monocytogenes in foods by immunomagnetic separation.

Immunomagnetic separation with immunomagnetic beads was used to isolate strains of Listeria monocytogenes both from pure cultures and from heterogeneous suspensions. The monoclonal antibodies used recognized all six strains of serotype 4 but only one of three strains of serotype 1. Coating procedure, incubation time, and number of immunomagnetic beads influenced the sensitivity of the isolation method. Less than 1 x 10(2) bacteria per ml in pure cultures and less than 2 x 10(2) bacteria per ml in enriched foods could be detected. The method represents a new approach to extraction and isolation of pathogenic bacteria directly from foods, after resuscitation, or from enrichment broths.

Agglutination Tests↗
PubMed1998

Isolation of Mycobacterium paratuberculosis from 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.

Animals↗
PubMed1998

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.

Adsorption↗
PubMed1998

Immunomagnetic separation with mediated flow injection analysis amperometric detection of viable Escherichia coli O157.

The coupling of an immunological separation (using immunomagnetic beads) with amperometric flow injection analysis detection of viable bacteria is presented. Using a solution containing Escherichia coli O157, the electrochemical response with two different mediators [potassium hexacyanoferrate(III) and 2,6-dichlorophenolindophenol] was evaluated in the FIA system. Antibody-derivatized Dynabeads were used to selectively separate E. coli O157 from a matrix. The kinetics and the capacity parameters regarding the attachment of bacteria to the immunobeads were studied. The immunomagnetic separation was then used in conjunction with electrochemical detection to measure the concentration of viable bacteria. A calibration curve of colony-forming units (cfu) against electrochemical response was obtained. The detection limit for this rapid microbiological method was 10(5) cfu mL-1, and the complete assay was performed in 2 h. Some advantages over ELISA methods are the direct detection of viable cells (and not total bacterial load) and the need for only one antibody (not enzyme-labeled), thus making the assay faster (only one washing step is necessary) and less expensive.

2,6-Dichloroindophenol↗
PubMed1996

Immunomagnetic separation can enrich fixed solid tumors for epithelial cells.

Immunomagnetic separation is a highly specific technique for the enrichment or isolation of cells from a variety of fresh tissues and microorganisms or molecules from suspensions. Because new techniques for molecular analysis of solid tumors are now applicable to fixed tissue but sometimes require or benefit from enrichment for tumor cells, we tested the efficacy of immunomagnetic separation for enriching fixed solid tumors for malignant epithelial cells. We applied it to two different tumors and fixation methods to separate neoplastic from non-neoplastic cells in primary colorectal cancers and metastatic breast cancers, and were able to enrich to a high degree of purity. Immunomagnetic separation was effective in unembedded fixed tissue as well as fixed paraffin-embedded tissue. The magnetically separated cells were amenable to fluorescence in situ hybridization and polymerase chain reaction amplification of their DNA with minimal additional manipulation. The high degree of enrichment achieved before amplification contributed to interpretation of loss of heterozygosity in metastatic breast cancers, and simplified fluorescence in situ hybridization analysis because only neoplastic cells were hybridized and counted. Immunomagnetic separation is effective for the enrichment of fixed solid tumors, can be performed with widely available commercial antibodies, and requires little specialized instrumentation. It can contribute to interpretation of results in situations where enrichment by other methods is difficult or not possible.

Breast Neoplasms↗
PubMed1995

Immunomagnetic separation of Salmonella from foods and their detection using immunomagnetic particle (IMP)-ELISA.

An immunomagnetic particle based ELISA (IMP-ELISA) for the detection of Salmonella from foods has been developed using Dynabeads anti-Salmonella (Dynal, Oslo, Norway). Appropriate sample preparation protocols to allow rapid detection of Salmonella serovariants in processed (powdered egg products) and non-processed (raw chicken) samples have been established. Pre-enriched broths of heat processed samples likely to harbour only low levels of competitive enteric flora, were boiled and used directly for IMP-ELISA. For non-heat processed or raw samples likely to contain higher numbers of such competing organisms, live Salmonella cells were first isolated by immunomagnetic separation (IMS) from standard pre-enrichment broths, and then post-selectively enriched for a short time in M-broth followed by boiling before IMP-ELISA. The total assay time including sample preparation was under 26 h for both types of procedure, with a lower detection limit of 10(5) Salmonella cells/ml of sample. In an evaluation of naturally contaminated poultry samples, all 45 of 48 samples previously shown to contain salmonellae in a comparison of ISO, IMS-Plating, Salmonella-Tek ELISA (Organon Teknika, Inc. Durham, NC) and a modification of the latter based on IMS, were identified as positive. None of the other methods gave positives for all 45.

Animals↗
PubMed1995

An evaluation of immunomagnetic separation for the detection of salmonellas in raw chicken carcasses.

Immunomagnetic separation techniques were used in the isolation of salmonella from raw chicken carcasses. Improved isolation rates were achieved with increased specificity and decreased processing time, although several technical difficulties remain to be addressed. Immunomagnetic separation offers significant potential for improvement on existing microbiological systems for the isolation of salmonella.

Animals↗
PubMed1996

The isolation and detection of Escherichia coli O157 by use of immunomagnetic separation and immunoassay procedures.

The use of immunomagnetic separation (IMS) techniques has been reported to reduce the total test time, and improve the sensitivity, of microbiological tests done on foods. This approach is being adopted in epidemiological investigations into suspected foodborne outbreaks of Escherichia coli O157 infection and has gained acceptance by public health laboratories and the food industry. This study demonstrated the ability of a commercially available IMS procedure, Dynabeads anti-E. coli O157, to enable detection of a few cells of E. coli O157 in 25 g of inoculated minced beef, giving results 1 d earlier than a cultural analysis of similar sensitivity. With correct choice of enrichment broths, IMS may increase isolation rate of E. coli O157 compared to that obtained using conventional cultural methods. It is suggested that this may be due to an increase in relative concentration of E. coli O157 compared with the background microflora present in minced beef, which may reduce reliability of non-IMS detection procedures by masking or mimicking target cells on selective/differential solid media. The use of an immunoassay incorporating an IMS step, EHEC-Tek (Organon-Teknika), enabled detection of a few cells of E. coli O157 in 25 g of minced beef. Comparison of the IMS-ELISA with a standard ELISA procedure (Tecra) indicated the sensitivity of the latter system to be greater, perhaps resulting in the higher isolation rate. The use of a method to reliability isolate and detect extremely low levels of E. coli O157 in a food is necessary to aid reduction in the incidence of this most serious of foodborne pathogens.

Animals↗
PubMed1997

Rapid purification of glial cells using immunomagnetic separation.

By purifying glial cells from brain tissue containing a heterogeneous cell population, a number of interactions that define glial cell diversification and function within the central nervous system have been determined. The current methods for purifying glial cells, however, can be time consuming and costly. In the following study we have adapted the technique of immunomagnetic separation to separately enrich 0-2A progenitor cells and astrocytes from the rat central nervous system (CNS). In this procedure, tissue from the CNS was enzymatically dissociated and incubated in a primary antibody specific to a surface antigen found on the target cell type (e.g. A2B5 or RAN-2). The target cells were then immunologically coupled to magnetic beads, which were precoated with a secondary antibody specific to the primary, and then separated out from the heterogeneous cell population using a magnetic field. We found that the immunomagnetic separation procedure, which was completed within 2 h, produced a near pure population of glial cells (> 99%). This was confirmed by the absence of unbound cells in the bead-bound fraction. The identification and viability of bead-bound cells were established by culturing these cells and subsequently examining their morphology and antigenic expression. This study shows that glial cell types can be separated out of brain tissue to near purity using immunomagnetic separation. This simple procedure is reliable, inexpensive, and achieves levels of purity and viability comparable with currently available techniques of immunopanning and fluorescence-activated cell sorting, within a fraction of the time.

Animals↗
PubMed1997

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.

Animals↗
PubMed1995

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.

Antigens, Viral↗
PubMed1997

An integrated system using immunomagnetic separation, polymerase chain reaction, and colorimetric detection for diagnosis of Plasmodium falciparum.

An integrated system for sample preparation and DNA detection of the malaria parasite using immunomagnetic separation in combination with the polymerase chain reaction (PCR) and colorimetric analysis is described. A cocktail of three monoclonal antibodies towards merozoite surface antigen-1 was used for magnetic capture of parasites from microliter amounts of whole blood. A sensitivity down to a parasitemia of 10(-6)% was achieved using cultured parasites as a model. The integrated approach was evaluated in a field study. A total of 410 blood samples from patients attending malaria clinics in Trat province and Kanchanaburi province in Thailand were analyzed. The samples were processed by immunomagnetic separation and transferred to central laboratory for PCR-based detection. Microscopic examinations on blood smears were done in parallel; 53% were positive using the DNA-based assay, while only 32% were positive using conventional microscopic analysis. This field study suggests a possible model for large-scale testing of malaria with an increased sensitivity compared with conventional methods.

Animals↗
PubMed1999

Evaluation of immunomagnetic separation and plating media for recovery of Salmonella from meat.

Immunomagnetic separation (IMS) was compared with selective enrichment in selenite cystine (SC) broth for isolation of Salmonella from 86 artificially contaminated ground beef samples. Both Rambach agar (RA) and Hektoen enteric (HE) agar were used as selective plating media. The highest count of Salmonella colonies per plate was obtained after enrichment in SC broth and plating on RA (mean value: 111.1+/-58.1 CFU per plate); the lowest count was obtained after IMS and plating on HE agar (mean value: 65.4+/-36.6 CFU per plate). Salmonella in preenrichment was concentrated 1.7-fold by IMS and represented 31% of the microbial population captured by the beads, but nonspecific binding was high. As a result of the large numbers of competing bacteria, isolations on both RA and HE agar following IMS were quite difficult (mean value for Salmonella colonies: 79.9+/-42.7 CFU per plate; mean value for non-Salmonella colonies: 144.1+/-75.7 CFU per plate; ratio of Salmonella to non-Salmonella colonies: 0.8). This study indicates that SC broth is superior to IMS in the isolation of Salmonella from raw ground meat.

Animals↗
PubMed1999

Rapid detection of Salmonella in chicken washes by immunomagnetic separation and flow cytometry.

Use of flow cytometry to rapidly detect Salmonella in chicken carcass washes was investigated. A direct immunomagnetic separation method was used to prepare samples and was found to be an effective method for separating target cells from debris in chicken carcass washes. When flow cytometry was combined with immunomagnetic separation, the average lowest detectable level of Salmonella detected was 2.3 x 10(4) CFU/ml. Fifty of 100 wash samples from six groups were inoculated with 2 x 10(-1) CFU of Salmonella Typhimurium per milliliter. After 18 h of enrichment at 37 degrees C, all samples were tested for Salmonella using flow cytometry and conventional culture methods. An identification correlation of 96% was found between flow cytometry and xylose-lysine-tergitol agar plating. Quantitative analysis established a significant linear relationship between the enumeration results of flow cytometry and xylose-lysine-tergitol agar plate counts (R2 = 0.796). Time required for flow cytometry, including sample processing and flow cytometric analysis, was less than 1 h.

Animals↗
PubMed1998

Immunomagnetic separation of Cryptosporidium parvum from source water samples of various turbidities.

Immunomagnetic separation (IMS) procedures which specifically capture Cryptosporidium oocysts and have the potential to isolate oocysts from debris have become commercially available. We compared two IMS kits (kit DB [Dynabeads anti-Cryptosporidium; product no. 730.01; Dynal A.S., Oslo, Norway] and kit IC1 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC, Portland, Maine]) and a modification of kit IC1 (kit IC2 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC]) at three turbidity levels (50, 500, and 5,000 nephelometric turbidity units [ntu]) by using water matrices obtained from different geographical locations. In deionized water, kit DB yielded recoveries between 68 and 83%, whereas the recoveries obtained with kits IC1 and IC2 were more variable and ranged from 0.2 to 74.5%. In water matrices with turbidity levels up to 500 ntu, the oocyst recoveries were more variable with kit DB; however, the recoveries were similar to those obtained in deionized water. In contrast, there were notable reductions in oocyst recoveries in the turbid matrices with kits IC1 and IC2, and the highest recovery (8.3%) was obtained with a 50-ntu sample. An examination of the effects of age on oocyst recovery with kit DB revealed that oocysts up to 16 weeks old yielded recoveries similar to the recoveries observed with fresh oocysts. These data indicate that all IMS kits do not perform equally well, and it is important to conduct in-house quality assurance work before a commercially available IMS kit is selected to replace flotation procedures for recovery of Cryptosporidium oocysts.

Animals↗
PubMed1994

Immunomagnetic separation of subpopulations of apolipoprotein A-I.

OBJECTIVE: This study was undertaken to measure the subfractions of high-density lipoprotein (HDL) in patients with diabetes or coronary artery disease and in normal control subjects. DESIGN: A new immunomagnetic separation technique was used to characterize the lipid profile in four groups: (1) control subjects, (2) patients with diabetes but no coronary artery disease (CAD), (3) those with CAD only, and (4) those with both diabetes and CAD. MATERIAL AND METHODS: To study the individual roles of the two discrete HDL subpopulations of particles--LpAI/AII (apolipoprotein [apo] A-I associated with A-II) and LpAI (apo A-I without A-II)--in lipoprotein metabolism, we developed an immunomagnetic separation technique using magnetic beads coated with antibodies to human apo A-II. The beads bind particles that contain both apo A-II and apo A-I and are precipitated by a magnetic field. LpAI levels were measured in the supernatant by performing an apo A-I radioimmunoassay. LpAI/AII levels were determined by subtracting the LpAI levels from total plasma apo A-I. RESULTS: In comparison with control subjects, patients with diabetes, CAD, or both had significantly decreased levels of LpAI/AII. LpAI levels were normal in patients with diabetes without CAD but significantly lower than control values in those with diabetes and CAD. CONCLUSION: Our findings suggest that both subpopulations of HDL particles have implications in the development of atherosclerosis in patients with and without diabetes.

Adult↗