Capturing B type acute lymphoblastic leukemia cells using two types of antibodies

One way to monitor minimal residual disease (MRD) is to screen cells for multiple surface markers using flow cytometry. In order to develop an alternative microfluidic based method, isolation of B type acute lymphoblastic cells using two types of antibodies should be investigated. The immunomagnetic beads coated with various antibodies are used to capture the B type acute lymphoblastic cells. Single beads, two types of beads and surface immobilized antibody were used to measure the capture efficiency. Both micro and nanosize immunomagnetic beads can be used to capture B type acute lymphoblastic cells with a minimum efficiency of 94% and maximum efficiency of 98%. Development of a microfluidic based biochip incorporating immunomagnetic beads and surface immobilized antibodies for monitoring MRD can be an alternative to current cost and time inefficient laboratory methods. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2737, 2019     

Production of and applications for a polyclonal IgY diagnostic reagent specific for <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis>

Antibodies specific to the cell surface antigens of Mycobacterium avium subsp. paratuberculosis (MAP) have multiple useful applications, e.g. organism detection, immunoconcentration, and cell visualization. The aim of this study was to produce and compare polyclonal antibodies for such research and diagnostic purposes. Three polyclonal antibodies to MAP were produced using sera from immunized rabbits and chickens plus naturally infected cows. Cross-reactive antibodies in each MAP antibody preparation were removed by absorption with heterologous mycobacterial and non-mycobacterial cells. The specificity of each resulting polyclonal antibody preparation was evaluated by ELISA to multiple bacterial cell wall extract antigens. After absorption, chicken anti-MAP IgY had the highest specificity of the three antibody preparations. FITC-la-beled anti-MAP IgY was used to effectively locate MAP in macrophages 12 h post-infection. Also, immunomagnetic beads coated with anti-MAP IgY enhanced recovery of MAP from bacterial suspensions in comparison with non-antibody coated beads. Anti-MAP IgY provides a novel new reagent with broad diagnostic and research applications requiring specific concentration, detection, and quantification of MAP.     

检测大肠杆菌(Escherichia coli) O157:H7免疫磁珠的制备、保存与应用

【背景】大肠杆菌(Escherichia coli) O157:H7是导致肠出血性大肠杆菌食源性疾病暴发的主要血清型,免疫磁珠(Immunomagnetic beads,IMBS)在E. coli O157的检测中发挥着重要作用,而免疫磁珠的稳定性、特异性、广谱性等性能指标关系着在实际应用中的使用效果。【目的】制备高效、稳定且具有广谱性的免疫磁珠,联合分子检测技术如环介导恒温扩增 (Loop-mediated isothermal amplification,LAMP)技术、PCR等,提高目标菌的检出率。【方法】采用新型的磁珠活化剂MIX&GO制备E. coli O157免疫磁珠,并进行广谱性以及特异性检测;针对6种试剂牛血清白蛋白(Bovine serum albumin,BSA)、酪蛋白(Casein)、海藻糖(Trehalose)、聚乙烯吡咯烷酮(Polyvinyl pyrrolidone,PVP)、抗坏血酸(Vitamin C)和防腐剂ProClin 300,利用正交试验L18(37)优化免疫磁珠保存液组分;采用IMBS-LAMP、IMBS-PCR、IMBS-生化、菌液-LAMP、菌液-PCR、显色平板-生化鉴定6种方式对20份生猪肉样品进行检测。【结果】利用MIX&GO活化剂制备的免疫磁珠捕获率最高达到81.5%±1.3%;免疫磁珠保存液最优配方为：牛血清白蛋白15.0 g/L,酪蛋白10.0 g/L,海藻糖10.0 g/L,PVP 2.0 g/L,抗坏血酸5.0 g/L,ProClin 300 2.5 g/L,保存6个月后免疫磁珠捕获率为75.5%;在20份生猪肉样品的检测中,自制磁珠和商品化磁珠与LAMP联用均检出9例阳性样品;IMBS-LAMP在6种检测方式中具有最高的检测灵敏度,但检出的样品会因磁珠抗体的差异而有所不同。【结论】与商品化磁珠相比,实验制备的免疫磁珠具有良好的特异性和广谱性,免疫磁珠-LAMP联用提高了目标菌的检出率,是一种高灵敏度、具有应用前景的检测方法。     

Fractionation of bovine spermatozoa for sex selection: A rapid immunomagnetic technique to remove spermatozoa that contain the H-Y antigen

A study was conducted to rapidly fractionate bovine spermatozoa on the basis of cell-surface H-Y antigen (i.e., Y chromosome-bearing spermatozoa). A novel, rapid immunomagnetic method was developed for removal of spermatozoa that bound to anti-H-Y IgG. Fluorescent labeling and flow cytometry were used to measure the efficiency with which spermatozoa binding to anti-H-Y were removed by the immunomagnetic technique. Washed bovine spermatozoa (n=7 bulls) were treated with a mouse monoclonal IgG antibody to H-Y antigen (MoAb 12/49). Fluorescent labeled goat antibody against mouse IgG was added to label those spermatozoa with cell-surface H-Y antigens. Supermagnetized polymer beads coated with an anti-antibody to the MoAb 12/49 were then added to the spermatozoa. After 20 min of incubation, spermatozoa were exposed for 2 min to a magnet, causing the magnetized particles to adhere to the sides of the tube. Nonmagnetized spermatozoa in the supernatent were aspirated and analyzed for fluorescent label by flow cytometry. Approximately 50% of spermatozoa not subjected to immunomagnetic separation were fluorescent labeled, and about one-half of the spermatozoa were observed microscopically to be bound to the magnetized polymer beads prior to magnetic separation (P<0.05). Following magnetic separation, only 1.2% (P<0.05) of the spermatozoa in the magnetic supernatent were fluorescent labeled. Assuming that only Y chromosome-bearing spermatozoa have cell-surface H-Y antigens, the present immunomagnetic fractionation removed almost all of the Y chromosome-bearing spermatozoa, leaving a population that was greater than 98% X chromosome-bearing spermatozoa.     

单增李斯特菌InternalinA的表达、纯化及多克隆抗体的制备

【目的】克隆表达单增李斯特菌膜表面蛋白InternalinA(InlA),经免疫家兔获得多克隆抗体,为建立其免疫磁珠富集快速检测方法奠定基础。【方法】利用生物软件设计单增李斯特菌inlA基因的引物,通过PCR扩增出inlA基因,并将其克隆至pET28a()原核表达载体,转化大肠杆菌BL21进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,ELISA分析其免疫原性。免疫家兔,制备其多克隆抗体。间接ELISA检测多抗的效价及交叉性,免疫荧光分析多抗与单增李斯特菌菌体结合的特异性。【结果】成功表达了InlA蛋白,融合表达产物分子量约为92 kD,质谱鉴定其为InlA蛋白;免疫家兔获得的抗血清效价为1:100 000,除与金黄色葡萄球菌约20%的交叉外,与副溶血弧菌等其它病源菌均无交叉;免疫荧光证实该多抗特异性结合于单增李斯特菌膜表面,与同种属的威尔斯李斯特菌不结合。【结论】成功制备了单增李斯特菌特异性的兔多克隆抗体,为单增李斯特菌免疫磁珠富集快速检测方法的建立奠定了基础。     

Tris(3-mercaptopropyl)-N-glycylaminomethane as a new linker to bridge antibody with metal particles for biological cell separations

Conjugates of nickel beads with CD8 and anti-red blood cell KC16 antibody were prepared by using the aminotrithiolate "spider" ligand, tris(3-mercaptopropyl)-N-glycylaminomethane, in its new function as a linker between the surface of nickel beads and antibody via activation of spider ligand attached to nickel beads with the common, heterobifunctional cross-linker, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). Raw nickel beads were cleaned by either mild sonication in a bath or by stronger probe sonication to remove surface nickel oxide layers, before attachment of the spider ligand. Scanning electron micrographs of the nickel beads before and after probe sonication showed a marked change from a corrugated to a smooth bead surface. Analyses of the supernatants of conjugation mixtures for antibody gave surface densities of 2.5-5.2 mg/m(2) for CD8 and 0.6-12 mg/m(2) for KC16 antibody runs. The antibody-spider-nickel bead conjugates were used in magnetic bead depletions of targeted CD8+ lymphocytes or red blood cells (rbcs) in whole blood of normal donors. For CD8 cell depletions, the undepleted controls and supernatants of depleted samples were analyzed for CD8/CD4 cell populations by flow cytometry with appropriate fluorescent antibody markers. Enumeration of red blood cells, white blood cells (wbcs), and platelets (plts) in undepleted controls and supernatants of depleted samples were carried out on appropriate hematology counters. Whole blood titer results with various lots of either CD8-spider-nickel or KC16-spider-nickel bead conjugates showed varying degrees of depletion ability as indicated by bead-to-cell ratios of 2-32 for CD8 beads and by rbc-to-bead ratios of 1.2-10 for KC16 beads. Moreover, varying degrees of specificity of CD8 beads for CD8+ cells over CD4+ cells and of KC16 beads for rbcs over white blood cells and platelets were observed from the normalized nontargeted cell population figures in undepleted controls versus supernatants of depleted samples.     

Development of monoclonal antibody-coated immunomagnetic beads for separation and detection of norovirus (genogroup II) in faecal extract samples

Aims:  The aim of this study is to develop an RT-PCR assay combined with immunomagnetic beads (IMS/RT-PCR) coating monoclonal antibody (Mab) for separation and detection of norovirus (genogroup II) in faecal samples. We furthermore compare its detection limits with IMS/RT-PCR using polyclonal antibody (Pab) and the TRIzol extraction method followed by RT-PCR (TRIzol-RT-PCR).
Methods and Results:  Mab-coated beads and Pab-coated beads were added to a series of tenfold dilutions of faecal extract containing norovirus in 1 ml PBS. After incubation and collection, the RNA was released by heating from virus separated by beads. The tenfold dilutions of faecal were also extracted with TRIzol reagent. The RNA was used as the template for RT-PCR detection (primers: JV12–JV13). IMS/RT-PCR using Mab showed an endpoint in the 10⁻⁷ dilution and was 10² times more sensitive than IMS/RT-PCR using Pab and was at least 10³ times more sensitive than TRIzol-RT-PCR method.
Conclusions:  IMS/RT-PCR using Mab proved to be a more sensitive method of noroviruses (NVs) detection than IMS/RT-PCR using Pab and the TRIzol-RT-PCR method.
Significance and Impact of the Study:  This is the first study to detect NVs with IMS/RT-PCR using Mab, and could serve as a model for future assays when broadly reactive NVs-specific Mabs are developed.     

Time-resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli O157:H7 in apple cider.

An immunoassay based on immunomagnetic separation and time-resolved fluorometry was developed for the detection of E. coli O157:H7 in apple cider. The time-resolved fluorescent immunoassay (TRFIA) uses a polyclonal antibody bound to immunomagnetic beads as the capture antibody and the same antibody labeled with europium as the detection antibody. Cell suspensions of 10(1) to 10(8) E. coli O157:H7 and K-12 organisms per ml were used to test the sensitivity and specificity of the assay. The sensitivity of the assay was 10(3) E. coli O157:H7 cells with no cross-reaction with K-12. Pure cultures of E. coli O157:H7 (10(1) to 10(5) CFU/ml) in apple cider could be detected within 6 h, including 4 h for incubation in modified EC broth with novobiocin and 2 h for the immunoassay. When apple cider was spiked with 1 to 10(3) CFU/ml of E. coli O157:H7 and 10(6) CFU/ml of K-12, our data show that the high level of K-12 in apple cider did not impede the detection of low levels of O157:H7. The minimum detectable numbers of cells present in the initial inoculum were 10(2) and 10(1) CFU/ml after 4- and 6-h enrichment. The TRFIA provides a rapid and sensitive means of detecting E. coli O157:H7 in apple cider.     

原核表达MurA蛋白并制备其多克隆抗体用于免疫磁珠快速检测单增李斯特菌

【目的】制备MurA多抗,结合免疫磁珠与选择平板进行单增李斯特菌的快速检测,建立单增李斯特菌的免疫磁珠快速检测方法。【方法】构建MurA的原核表达载体,转化大肠杆菌进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,再免疫小鼠,制备其多克隆抗体。用所获多抗制备免疫磁珠,建立单增李斯特菌免疫磁珠-选择性培养基检测方法,并对人工污染牛奶样品进行检测。【结果】在大肠杆菌中高效表达了分子量约为72 kD的可溶性融合蛋白,质谱鉴定其为MurA蛋白;免疫小鼠获得的抗血清效价达1:10 000,与伤寒沙门氏菌、副溶血弧菌、大肠杆菌及属内其它病原菌均无交叉;所建立的免疫磁珠-选择性培养基检测法可检出浓度为103 CFU/mL及以上的单增李斯特菌,仅与英诺克李斯特菌存在一定交叉反应;牛奶样品单次仅需9 h增菌就能被检出,较常规增菌时间缩短39 h;检测限为0.4 CFU/mL。【结论】表达并纯化得到高纯度的单增李斯特菌MurA蛋白,制备的鼠源多克隆抗体亲和力高,特异性好;建立了快速检测单增李斯特菌的免疫磁珠联合选择性培养基法,在灵敏度不变的情况下,实现24 h内成功对牛奶样品的检测,较国标法减少42 h以上。     

Sensitive detection of Escherichia coli O157:H7 in food and water by immunomagnetic separation and solid-phase laser cytometry

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r2 = 0. 95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r2 = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.     

免疫磁珠技术分离唾液A/B血型抗原并用于吸附血清A/B抗体

目的:应用免疫磁珠分离技术获得具有良好抗原性的A/B血型抗原,并探究其作为ABO血型抗体吸附剂去除A/B抗体的可行性。方法:将含有血型物质的唾液进行预处理,再与包被了抗体的磁珠混合,分离出纯度较高的A/B抗原,运用酶联免疫及凝集抑制试验验证所得抗原的抗原性及是否存在交叉反应。用未纯化A/B抗原和纯化A/B抗原包被磁珠,对含有抗A/B IgM、IgG的血清进行抗体吸附,用纯化A/B抗原对100份来自O型血孕妇的临床血清样本进行抗体吸附,分别评价其吸附效果。结果:纯化抗原与对应抗体反应后,其吸光度显著高于对照组(A抗原与A抗体0.85±0.12 vs.0.27±0.03,P0.01;B抗原与B抗体0.86±0.09 vs.0.24±0.06,P0.01),与其它类型抗体反应后的吸光度值与对照组比较差异无统计学意义(P0.05)。进行红细胞凝集抑制试验时,纯化抗原可显著抑制相应抗体与红细胞的凝集反应,对其它类型抗体与红细胞的凝集没有抑制作用。血清抗体吸附实验表明纯化抗原的吸附效率比未纯化抗原的高(97.00%vs.88.00%,P0.001)。临床样本抗体吸附实验显示,纯化A抗原对抗A IgM/IgG的吸附效率分别为96.88%、98.44%;纯化B抗原对抗B IgM/IgG的吸附效率分别为96.88%、98.44%。结论:磁珠纯化抗原能特异性地与对应抗体结合,有效吸附血清中的血型抗体,有望作为合成A/B抗原的替代品。     

Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation

The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.     

Development of an immunomagnetic bead-immunoliposome fluorescence assay for rapid detection of Escherichia coli O157:H7 in aqueous samples and comparison of the assay with a standard microbiological method

The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic bead complexes were isolated by magnetic separation, washed, and incubated with sulforhodamine B-containing immunoliposomes specific for E. coli O157:H7; the final immunomagnetic bead-E. coli O157:H7-immunoliposome complexes were again isolated by magnetic separation, washed, and lysed with a n-octyl-beta-d-glucopyranoside to release sulforhodamine B. The final protocol took less than 8 h to complete and had a detection limit of less than 1 CFU of E. coli O157:H7 per ml in various aqueous matrices, including apple juice and cider. To validate the protocol at an independent facility, 100-ml samples of groundwater with and without E. coli O157:H7 (15 CFU) were analyzed by a public health laboratory using the optimized protocol and a standard microbiological method. While the IMB/IL fluorescence assay was able to identify E. coli O157:H7-containing samples with 100% accuracy, the standard microbiological method was unable to distinguish E. coli O157:H7-spiked samples from negative controls without further extensive workup. These results demonstrate the feasibility of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of E. coli O157:H7 in aqueous samples.     

Immunomagnetic separation of scum-forming bacteria using polyclonal antibody that recognizes mycolic acids

Mycolic acid-containing bacteria (mycolata) are thought to be involved in scum formation in aeration basins of activated sludge plants due to their ability to produce biosurfactants and their cell surface hydrophobicity. To isolate these bacteria, immunomagnetic separation (IMS) using an anti-mycolic acid polyclonal antibody was investigated. IMS that targeted Gordonia amarae SC1 exhibited a 100% recovery at 5x10(3) CFU ml(-1). At cell concentration of 7.8x10(6) CFU ml(-1), the recovery was lowered, but 80% of cells were still captured. Effect of bead concentrations on the recovery of SC1 at 10(6) CFU ml(-1) was examined. The results showed that addition of more than 6-7x10(6) beads for 1x10(6) CFU reached a maximum recovery (83%). Furthermore, the IMS procedure optimized with SC1 cells was tested with another mycolata. The results suggested that variation of the recovery for each mycolata is dependent on the specificity of the polyclonal antibody and that mycolata which are recognized by the antibody can be recovered by this procedure.     

Affinity binding of cells to cryogel adsorbents with immobilized specific ligands: effect of ligand coupling and matrix architecture

The capture of human acute myeloid leukemia KG-1 cells expressing the CD34 surface antigen and the fractionation of human blood lymphocytes were evaluated on polyvinyl alcohol (PVA)-cryogel beads and dimethyl acrylamide (DMAAm) monolithic cryogel with immobilized protein A. The affinity ligand (protein A) was chemically coupled to the reactive PVA-cryogel beads and epoxy-derivatized monolithic cryogels through different immobilization techniques and the binding efficiency of the cell surface receptors specific antibody-labeled cells to the gels/beads was determined. The binding of cells to monolithic cryogel was higher (90-95%) compared with cryogel beads (76%). B-lymphocytes, which bound to the protein A-cryogel beads, were separated from T-lymphocytes with yields for the two cell types 74 and 85%, respectively. About 91% of the bound B-cells could be recovered without significantly impairing their viability. Our results show differences in the percentage of cell-binding to the immunosorbents caused by ligand density, flow shear forces and bond strength between the cells and the affinity surface once distinct chemical coupling of protein A, size of beads, sequence of antibody binding to protein A adsorbents, morphology and geometry of surface matrices were compared.     

Immunomagnetic detection of Bacillus stearothermophilus spores in food and environmental samples.

There are currently no methods for the rapid and sensitive detection of bacterial spores that could be used to direct raw materials containing high spore loads away from products that pose a food safety risk. Existing methods require an overnight incubation, cannot detect spores below 10(5) CFU/ml, or are not specific to particular species. This work describes a method to specifically detect < 10(4) CFU of bacterial spores per ml within 2 h. Polyclonal antibodies to Bacillus stearothermophilus spores were attached to 2.8-micron-diameter magnetic polystyrene beads by using a polythreonine cross-linker via the antibody carbohydrate moiety. A biotin-avidin-amplified sandwich enzyme-linked immunosorbent assay coupled to a fluorescent substrate was used to quantitate captured spores. The concentration of B. stearothermophilus spores in samples was linearly correlated to fluorescent activity (r2 = 0.99) with a lower detection limit of 8 x 10(3) CFU/ml and an upper detection limit of 8 x 10(5) CFU/ml. The detection limits are not fixed and can be changed by varying the immunomagnetic bead concentration. Several food and environmental samples were tested to demonstrate the versatility of the assay.     

Capture antibody targeted fluorescence in situ hybridization (CAT-FISH): dual labeling allows for increased specificity in complex samples

Pathogen detection using biosensors is commonly limited due to the need for sensitivity and specificity in detecting targets within mixed populations. These issues were addressed through development of a dual labeling method that allows for both liquid-phase fluorescence in situ hybridization (FISH) and capture antibody targeted detection (CAT-FISH). CAT-FISH was developed using Escherichia coli O157:H7 and Staphylococcus aureus as representative bacteria, and processing techniques were evaluated with regard to FISH intensities and antibody recognition. The alternative fixative solution, methacarn, proved to be superior to standard solid-phase paraformaldehyde fixation procedures, allowing both FISH labeling and antibody recognition. CAT-FISH treated cells were successfully labeled with FISH probes, captured by immunomagnetic separation using fluorescent cytometric array beads, and detected using a cytometric array biosensor. CAT-FISH treated cells were detectable with LODs comparable to the standard antibody-based technique, (~ 10³ cells/ml in PBS), and the technique was also successfully applied to two complex matrices. Although immunomagnetic capture and detection using cytometric arrays were demonstrated, CAT-FISH is readily applicable to any antibody-based fluorescence detection platform, and further optimization for sensitivity is possible via inclusion of fluorescently tagged antibodies. Since the confidence level needed for positive identification of a detected target is often paramount, CAT-FISH was developed to allow two separate levels of specificity, namely nucleic acid and protein signatures. With proper selection of FISH probes and capture antibodies, CAT-FISH may be used to provide rapid detection of target pathogens from within complex matrices with high levels of confidence.     

Specific detection of cytopathogenic Listeria monocytogenes using a two-step method of immunoseparation and cytotoxicity analysis

The development of rapid methods for detection of viable Listeria monocytogenes is crucial to prevent listeriosis and product recalls. While immunomagnetic separation has been used for isolating Listeria spp., lack of specificity and pathogenicity determination render this method unsatisfactory. A two-step method using Protein A agarose beads (Immunobeads) coated with a more specific antibody, monoclonal antibody (MAb)-C11E9 for L. monocytogenes was developed. Immunobeads were allowed to capture Listeria cells from a variety of samples and tested for cytopathogenic action on a murine hybridoma B-lymphocyte, Ped-2E9 cell line by Trypan blue staining, and by an alkaline phosphatase (AP)-based cytotoxicity assay. The two-step method was used to test uninoculated hotdogs, bologna, and raw beef, chicken, and pork samples, following selective enrichment in half-Fraser broth. Pure culture studies proved the assay to be specific for L. monocytogenes, while a similar assay with Dynal Anti-Listeria immunomagnetic beads was positive for L. monocytogenes, L. ivanovii, and L. seeligeri. Detection and confirmation of cytopathogenicity of Listeria cells from food samples after 24-h selective enrichment were completed in 2-4 h. Isolates were further analyzed by the CAMP test for hemolytic activity and RiboPrinter for genomic patterns. Using immunoseparation and cytotoxicity as a two-step rapid method, viable L. monocytogenes could be isolated, detected, and confirmed as cytopathogenic in 28 h or less.