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PubMed1997

Isolation of proximal and distal tubule cells from human kidney by immunomagnetic separation. Technical note.

After collagenase digestion and Percoll density gradient centrifugation of human renal tissue, tubular epithelial cells of the proximal and the distal segments were isolated with an immunomagnetic method using MACS microbeads. To enrich proximal tubular (PT) cells we used a monoclonal antibody (mAb) against aminopeptidase M (APM, CD 13), specific of the proximal tubule. Distal tubular (DT) cells were isolated through a mAb recognizing Tamm-Horsfall glycoprotein (THG), a specific antigen for the thick ascending limb and the early distal convoluted tubule. Cells of the proximal primary isolate were histochemically strongly positive for aminopeptidase M (98.6%), however, cells of the distal portion were negative (98.7%). Ultrastructural analysis of PTC primary isolates revealed highly preserved brush border microvilli, well-developed endocytosis apparati and numerous mitochondria, whereas DTC primary isolates showed smaller cells with basolateral invaginations and less apical microvilli. Characterization by immunofluorescence indicated the coexpression of cytokeratin and vimentin, whereas staining for desmin, smooth muscle actin, a fibroblast-specific marker and von Willebrand factor was negative. Cultured PT and DT cells displayed different adenylate cyclase responsiveness to hormonal stimulation. PTH (10(-6) M) increased cAMP production in distal cells up to 32.8-fold of the basal level and in proximal only up to 3.5-fold (10(-8) M, DT 14.4x and PT 2.25x). Calcitonin stimulated adenylate cyclase in DT in a dose dependent fashion (10(-6) M, 4.3x; 10(-8) M, 2.25x), whereas only a low calcitonin response was found in PT cells (10(-6) M, 1.6x; 10(-8) M, 1.4x). AVP (10(-6) M) activated the distal cAMP-production only up to 1.9x of the basal level, but the proximal cAMP-production was negligible (only 1.3x the basal level). The data of this study indicate the proximal and distal tubule origin of the cultured cells that were isolated according to their segment-specific antigens.

Animals↗
PubMed1997

Detection of Campylobacter jejuni in food and poultry viscera using immunomagnetic separation and microtitre hybridization.

Thermophillic Campylobacter and Camp. jejuni were detected from samples of chicken liver, gall bladder, muscle and contaminated milk and chicken meat after an enrichment step by using immunomagnetic capture of cells with monoclonal antibody against a specific outer membrane protein of thermophilic Campylobacter. The detection of captured cells was achieved using two different hybridization methods. In one of the methods, the captured cells were lysed by guanidine isothiocyanate and the 23S rRNA was reacted with a microtitre plate-immobilized rDNA probe specific for thermophilic Campylobacter. In the other method, the captured cells were subjected to lysis by ultrasonication and the genomic DNA reacted with a microtitre plate-immobilized RNA probe specific for Camp.jejuni. Detection of the RNA-DNA hybrids formed in the wells was carried out using a monoclonal anti-RNA-DNA hybrid antibody.

Animals↗
PubMed1993

Cytokeratin profile of immunomagnetically separated epithelial subsets of the human mammary gland.

We have used enzymatic and immunomagnetic techniques for the physical separation of the basal and luminal epithelium of the human mammary gland. Immediately after tissue dissociation and cell sorting, we have examined the steady-state CK composition of these cells individually by direct two-dimensional gel electrophoresis of intermediate filament extracts. Our results demonstrate that cytokeratins typical of simple and stratified epithelial cells are simultaneously expressed by both mammary epithelial subclasses in vivo. Moreover, the entire spectrum of cytokeratins seen in vivo is also maintained in short-term cultures of human mammary epithelium. A comparison of the data obtained by direct cytokeratin analysis with published indirect immunolocalization studies is presented. The ability to isolate purified epithelial subsets from normal human breast tissue by a simple immunomagnetic procedure demonstrated here can facilitate the development of relevant model systems for studying the regulatory components in growth, differentiation and malignant transformation of the human breast.

Antibodies, Monoclonal↗
PubMed1995

Isolation of pure populations of epithelial and myoepithelial cells from the normal human mammary gland using immunomagnetic separation with Dynabeads.

Monoclonal antibodies to epithelial membrane antigen and common acute lymphoblastic leukemia antigen were bound to second antibody-coated magnetic microspheres. These specific antibody/bead complexes were then used to directly isolate purified epithelial and myoepithelial cells from normal breast organoid cell preparations by magnetic separation. Near homogeneous cell populations were selected with yields of 10-20 x 10(6) epithelial and myoepithelial cells per organoid preparation. Repeated purification steps allowed almost complete depletion of myoepithelial cells for RNA studies. Tissue culture of separated cell populations in appropriate defined media further ensured purity of cell type and was unimpeded by Dynabead attachment.

Antibodies, Monoclonal↗
PubMed2006

Rat liver endothelial cells isolated by anti-CD31 immunomagnetic separation lack fenestrae and sieve plates.

The gold standard for the identification of sinusoidal endothelial cells (SEC) is the presence of fenestrae organized in sieve plates, which is characteristic of SEC in vivo. One of the methods currently in use to isolate SEC is immunomagnetic sorting for CD31. However, there is evidence to suggest that CD31 is not present on the surface of differentiated SEC. The present study used scanning electron microscopy to image rat hepatic endothelial cells isolated by anti-CD31 and immunomagnetic sorting and cells isolated by gradient centrifugation and centrifugal elutriation. Cells isolated by elutriation had well-developed fenestrae and sieve plates, whereas cells isolated by anti-CD31 and immunomagnetic sorting had significantly fewer fenestrae organized in sieve plates. In conclusion, cells isolated by anti-CD31 and immunomagnetic sorting lacked the hallmark features of SEC.

Animals↗
PubMed1996

A comparison of immunomagnetic separation, direct culture and polymerase chain reaction for the detection of verocytotoxin-producing Escherichia coli O157 in human faeces.

Verocytotoxin-producing Escherichia coli O157 (O157 VTEC) has become well recognized as an important enteric pathogen. The number of organisms present in environmental and clinical samples may be low and efforts have been made to increase the sensitivity of O157 VTEC detection. Immunomagnetic seperation (IMS) has been shown to improve O157 VTEC detection in bovine faeces and food samples. A milkborne outbreak of O157 VTEC infection allowed us to compare the isolation rates from human faeces by IMS, direct faecal culture on sorbitol-MacConkey agar and a PCR test for verotoxin gene carriage. Of 142 faecal samples examined, 20 were positive on both direct culture and IMS and a further 13 on IMS alone. Therefore, IMS increased the detection rate of individual cases of O157 VTEC infection and also compared well with PCR. We recommend IMS for use in routine diagnostic laboratories where a more sensitive method than direct faecal culture is required for O157 VTEC isolation.

Animals↗
PubMed1993

Lack of IL-1 secretion from human myeloma cells highly purified by immunomagnetic separation.

This paper describes a method for purification of human myeloma cells. Mononuclear cells from six bone marrow samples and one pleural fluid sample from multiple myeloma patients were incubated with B-B4, a monoclonal antibody that is specific for plasma cells, and the B-B4+ cells were isolated using monosized magnetic beads coated with sheep anti-mouse Ig. With this positive selection method it was possible to achieve primary cultures with more than 99% myeloma cells. The average viability of these cultures was 81%. The B-B4 antibody did not alter proliferation or cytokine production of the myeloma cell lines U-266 and JJN-3. The B-B4+ myeloma cultures did not produce IL-1 and made only small amounts of IL-6 (< 93 pg/ml), whereas the cells remaining after extraction of the B-B4+ cells produced IL-1 (89-350 pg/ml) and large amounts of IL-6 (520-17,000 pg/ml). This indicates that the B-B4+ myeloma cells are not directly responsible for the overproduction of these cytokines in multiple myeloma. This separation technique gave higher purity of myeloma cells than has been previously reported for any negative selection method and is recommended when high culture purity is of critical interest.

Adult↗
PubMed1999

Immunomagnetic separation for enrichment and sensitive detection of disseminated tumour cells in patients with head and neck SCC.

Screening for malignant cells in the blood and bone marrow was introduced as a strategy for the improved detection of tumour spread and may predict the development of distant metastases. The sensitivity of these approaches depends on several factors, including the choice of antibody for immunocytochemistry (ICC) and the number of cells examined. In this study criteria have been defined for scoring cells reactive with a pan-cytokeratin antibody as tumour, by comparing immunostained cells in clinical samples obtained from head and neck cancer patients and a control group without epithelial malignancy. When leucocyte subfractions are prepared by density gradient separation (DGS) from central venous blood obtained from patients with advanced head and neck squamous cell carcinoma (SCC) and screened by ICC, epithelial tumour cells sediment preferentially with the mononuclear cells but may also be detected in the granulocyte (GC) fraction. Some cases were found to have more tumour cells in the GC fraction. Similar results were seen in model experiments. To increase the sensitivity of the ICC approach, the efficiency of positive immunomagnetic selection (IMS) using Dynabeads coated with an antibody recognizing the Ber-EP4 epitope has been compared with negative IMS using anti-CD45 Dynabeads. Tumour cells were recovered from bone marrow aspirates for 2/17 cases using the positive enrichment technique and for 11/17 patients following negative IMS. These findings justify prospective studies incorporating negative IMS to establish the prognostic significance of these disseminated tumour cells for this group of patients.

Antigens, Neoplasm↗
PubMed2005

Detection of viable Salmonella using microelectrode-based capacitance measurement coupled with immunomagnetic separation.

In this study, we demonstrated the use of a general medium--brain heart infusion (BHI) broth that is not specifically formulated for impedance measurement, to achieve detectable impedance signals by using an interdigitated microelectrode (IME) with capacitance measurement at low frequencies. Anti-Salmonella antibody coated immunomagnetic beads were used to separate S. typhimurium from samples to provide the selectivity to this method. From analysis based on the equivalent circuit of the IME system, we found that the impedance change in BHI broth resulting from the growth of Salmonella was indeed the change in the double layer capacitance and could be monitored at 10 Hz using the IME. The results indicated that medium modification to improve impedance signal is not necessary with this IME system. However, effective immunological separation for the target organism is required for the selectivity when non-selective media are used. This finding provides a more flexible option of medium in impedance methods, which may provide opportunities to test those species of bacteria that have no suitable conductance growth medium. The detection time, t(d), was obtained from the impedance growth curve (impedance against bacterial growth time) at 10 Hz at the point where the impedance started to change. A linear relationship between the detection time and the logarithmic value of the initial cell number (N) was found in the Salmonella cell number ranging from 10(1) to 10(6) cfu/ml. The regression equation was t(d) = -1.22Log N + 8.90, with R2 = 0.95. The detection times for the initial cell number of 10(1) CFU/ml and 10(6) CFU/ml are 8 h and 1.5 h, respectively. This method is more sensitive than impedance methods using conventional electrodes.

Biosensing Techniques↗
PubMed1997

Immunomagnetic separation and PCR for detection of Mycobacterium ulcerans.

We have developed a technique based on the use of monodisperse magnetic beads to isolate Mycobacterium ulcerans from heterogenous mixtures, prior to PCR amplification. Using this method, we were able to detect M. ulcerans in water samples taken from Phillip Island, Australia, the site of several outbreaks of M. ulcerans disease in recent times.

Antibodies, Bacterial↗