Multi-enzyme isothermal rapid amplification assay for the detection of Escherichia coli O157∶H7 / 上海预防医学

ObjectiveA rapid enrichment and detection method for Escherichia coli O157∶H7 was developed by using multienzyme isothermal rapid amplification （MIRA） fluorescence method combined with metal organic frameworks immunomagnetic beads. MethodsUsing rfbE gene as the target， the primers， probes and reaction system were screened， and the specificity， sensitivity and practical application of this method were investigated. ResultsThe detection limit of Escherichia coli O157∶H7 was 1.18×105 CFU‧mL-1， and the detection limit of DNA concentration was 9 pg‧μL-1. The detection process was completed in 20 minutes. The test results of 47 strains （24 target strains and 23 non-target strains） were consistent with real-time PCR （RT-PCR）. ConclusionA method based on metal-organic framework immunomagnetic beads enrichment combined with MIRA assay is developed in this study. The method is simple， rapid and suitable for rapid enrichment and detection of Escherichia coli O157∶H7 in food.

Efficacy of folate-coupled quantum dots immunomagnetic beads method for detecting circulating tumor cells in epithelial ovarian cancer and the association of circulating tumor cells with clinicopathological features / 肿瘤研究与临床

Objective:To explore the effectiveness of folate-coupled quantum dots (FA-QD) immunomagnetic beads method for detecting circulating tumor cells (CTC) in epithelial ovarian cancer and the association of CTC with clinicopathological features of tumor patients.Methods:A total of 67 ovarian cancer patients in Shanxi Provincial People's Hospital from August 2019 to January 2020 were selected. Ovarian cancer SKOV-3 cells were divided into 5 cell number gradients (0, 100, 150, 200, 300), the detection rates of CTC were compared by using epithelial cell adhesion molecule (EpCAM) immunomagnetic beads (single standard method) and FA-QD immunomagnetic beads method (double standard method). The number of CTC in peripheral blood of ovarian cancer patients was detected by using FA-QD immunomagnetic beads method, and those with positive CTC under fluorescence microscope were treated as CTC positive patients. The association of CTC with clinicopathological factors and tumor markers of tumor patients was analyzed.Results:The average capture efficiency rate of CTC in SKOV-3 cells detected by FA-QD immunomagnetic beads method (83.4%) was higher than that by EpCAM immunomagnetic beads method (70.3%). Among 67 patients of ovarian cancer, the proportion of CTC positive patients was 30.0% (3/10) in stage Ⅰ-Ⅱ, 91.9% (34/37) in stage Ⅲ, 95.0% (19/20) in stage Ⅳ, and the difference was statistically significant ( P < 0.05). The proportion of CTC positive patients with lymph node metastasis was higher than that of patients without lymph node metastasis [97.1% (33/34) vs. 69.7% (23/33)], and the difference was statistically significant ( P < 0.05). The proportion of CTC positive patients with human epididymis protein 4 (HE4)>110 pmol/L was lower than that of patients with HE4 ≤ 110 pmol/L [58.8% (10/17) vs. 92.0% (46/50)], and the difference was statistically significant ( P = 0.005). There were no statistically significant differences in the proportion of CTC positive patients stratified by age, menopause, pathological differentiation, distant metastasis, carbohydrate antigen (CA) 125, CA199, carcino-embryonic antigen (CEA) (all P > 0.05). Conclusions:FA-QD immunomagnetic beads method can effectively detect CTC in peripheral blood of patients with epithelial ovarian cancer. The level of CTC in patients with epithelial ovarian cancer is related to lymph node metastasis, clinical TNM stage and HE4 level.

Evaluation of a novel immuno-magnetic assay technology for rapid detection of dengue NS1 antigen

Background: Serological diagnosis of dengue fever is based on the detection of NS1 antigen and further confirmed by the assay of dengue IgM and IgG. Rapid Diagnostic Tests (RDTs) are no longer recommended for use by the Government of India. However, there is a requirement of rapid results without compromising on the test quality. Accordingly, we undertook an evaluation of a novel cartridge-based system employing a unique table-top diagnostic device for dengue NS1 antigen by BluSense Diagnostics incorporating a patented Immuno-Magnetic Assay (IMA).Methods: A total of 309 samples were tested on unlinked anonymous basis. Each sample was tested using NS1 Ag ELISA (Microlisa, J. Mitra), ViroTrack and NS1 RDT (SD Alere) and results were recorded. Discordant samples were further tested by dengue NS1 AG ELISA (Panbio) and dengue IgG/IgM Maclisa (J. Mitra).Results: When compared with approved ELISA kits used globally the Virotrack test returned a sensitivity of 94.74%; specificity of 97.44%; PPV of 97.30%; NPV of 95% and accuracy of 96.10%. Hourly throughput is 5-6 samples.Conclusions: The Virotrack system is highly suitable as a POCT module in HCFs with low to moderate workload that employs novel technology, is rapid, user friendly and comparable to the ELISA in sensitivity and specificity. The equipment is user friendly and can work both on plasma/serum and whole blood. Model with multiple modules (3-4) will improve the throughput and turnaround time. Evaluation of this novel technology has been done in India for the first time.

Isolation and Biological Characterization of Lung Cancer Stem Cells / 中国医学科学院学报

To explore the methods of screening and biological characteristics of lung cancer stem cells. We selected the ABCG2 and ABCG2 cells from SPC-A-1/adriamycin(ADM)cell line induced by ADM and analyzed the tumorigenicity of ABCG2 and ABCG2 cells by flow cytometry and transplantation in nude mice. The average fluorescence intensity of SPC-A-1 cells was(1.001±0.014)×10 ,which was significantly lower than that of SPC-A-1/ADM cells [(10.257±0.023) ×10 ](=17.320,=0.001);the difference was also statistically significant between the ABCG2/BCRP-FITC treatment group and the SPC-A-1 control group(=5.269,=0.021) and the SPC-A-1 control group(=6.869, =0.012) and between the SPC-A-1/ADM cell control group and the SPC-A-1/ADM cell homotype control group(=8.112,=0.015).The positive rate of SPC-A-1/ADM cells treated with ABCG2/BCRP-FITC was 9.8%,39.84 times higher than that of SPC-A-1 cells;it showed significant difference between the ABCG2/BCRP-FITC group and the SPC-A-1/ADM group(=9.120,=0.005) and the SPC-A-1/ADM group(=8.257,= 0.006).The positive rate of group B cells was 684 times that of group A cells,and the difference was statistically significant(=11.235,=0.001),and the fluorescence intensity of group B cells was strong.The average tumorigenic volume of the mice inoculated with SPC-A-1 cells,group A cells,and group B cells was(6.96±1.82),(6.70±2.55),and(9.17±2.41) mm ,respectively.Among them,group B was the highest,but there was no significant difference among these three groups(=2.362,=0.086).The tumorigenic rate of group B cells was 75.00%,which was significantly higher than that of SPC-A-1 cells and group A cells(=19.780,=0.002). ABCG2 cells from human lung adenocarcinoma SPC-A-1/ADM cell line can be isolated by ABCG2 antibody combined with immunomagnetic beads sorting method,and the tumor formation rate in nude mice can be observed to explore the identification and biological characterization of lung cancer stem cells.

Separation and forensic identification of sperm from cell mixtures using anti-hLCN6 monoclonal antibody coupled magnetic beads / 生物工程学报

Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10⁻⁹ mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 10³/mL and 100% when the sperm cells number was equal to or more than 10⁴/mL. When the number of sperm cells was 10³/mL, 10⁴/mL and 10⁵/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.

Primary culture of neurons under low density conditions by immunomagnetic beads method combined with conditioned medium / 中国药理学与毒理学杂志

OBJECTIVE To establish a primary culture of neurons under low density conditions by immunomagnetic beads (1MB) based cell separation technology combined with astrocyte conditioned medium (A-CM). METHODS The cerebral hemispheres of neonatal KM mice within 24 h of birth were mechanically isolated and trypsin digested to prepare cell suspension for primary culture. After 10 d of culture, the microglia and other irrelevant cells were removed by constant temperature oscillation, while the purity of astrocytes was identified by immunofluorescence staining with anti-glial fibrillary acidic protein (GFAP) antibody. A-CM was prepared using cell cultures that met purity requirements. The cerebral cortex and hippocampus of neonatal KM mice within 24 h of birth were mechanically isolated and trypsin digested to prepare cell suspension. High-purity neurons were obtained by immunomagnetic beads based cell separation technology, and A-CM was applied to primary culture of neurons under low density conditions. Morphological parameters of neurons during culture were observed, and neurons were identified by immunofluorescence staining with anti-160 ku neurofilament antibody. RESULTS The purity of astrocytes was over 95% by cellular immunofluorescence staining identification, which met the cell purity requirements of subsequent experiments. Morphological observation and cellular immunofluorescence staining of the mouse cortex and hippocampal neurons showed that the neurons grew well under low density culture conditions with high purity, and the morphological characteristics were distinct at different developmental stages. CONCLUSION The primary culture of the mouse cerebral cortex and hippocampal neurons under low density conditions is successfully established.

Analysis of Detection of Mixed Semen Stains with Different Immunological Test Methods and Case Applications / 法医学杂志

Objective To perform the separation and confirmation of mixed semen stains with immunological test method, and find a more effective method for the detection of mixed semen stains. Methods The semens of three volunteers were mixed. The mixed semen stains were processed and tested with prostate-specific antigen （PSA） colloidal gold immunoassay strip method, immunomagnetic beads method and laser capture microdissection, respectively. Statistics of the results of STR were gathered and compared with those of a single semen stain. Results After PSA colloidal gold immunoassay strip method testing, the samples showed a purplish red line in the test area and the control area. The results obtained with the immunomagnetic beads method showed a more complete and effective short tandem repeat （STR） sequence. The mixed semen stains were processed with laser capture microdissection and low volume amplified. The results were summarized and superimposed to obtain a complete single typing, which matched the single semen stain typing, with a typing success rate of 84.00%. Single suspect Y-STR typing was obtained with the application of the method above in actual cases, which provided evidence basis for rapid solving of the case. Conclusion The combination of PSA colloidal gold immunoassay strip method, immunomagnetic beads method and laser capture microdissection can be used to separate and confirm the mixed semen stains.

Comparison of three diagnostic methods for Salmonella enterica serovars detection in chicken rinse / Comparação de três métodos diagnósticos para detecção de Salmonella enterica em lavados de carcaça de frango

Salmonella detection is a key point in food safety testing, because of the frequent association of this pathogen with food poisoning in humans. The standard bacteriological tests currently used for Salmonella-detection are time-consuming; therefore, there is a need to develop alternative methods to accelerate the detection. In order to accelerate Salmonella diagnosis, we used the immunomagnetic separation assay associated with bacteriophage P22 for the rapid detection of the following Salmonella serovars in chicken rinses of drumsticks, artificially contaminated with 5, 10, and 100 CFU/25mL of bacteria: Salmonella enterica subsp. enterica serovar Heidelberg (S. Heidelberg), Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium). The efficiency of the technique, represented by the time required for detection of positive and negative samples, was compared with that of the standard diagnostic tests used for this pathogen, the bacteriological assay and the polymerase chain reaction (PCR)-based test. This study confirmed the ability of the bacteriophage-associated immunomagnetic separation assay to identify 99.6% of Salmonella-positive samples of the three serovars tested. In contrast, the bacteriological assay and PCR-based test detected 95.1% and 98.5% of the Salmonella-positive samples respectively.(AU)

A detecção de Salmonella é um ponto crucial para a segurança alimentar, devido a frequente associação deste patógeno com infecções alimentares em humanos. O método padrão para detecção de Salmonella é o bacteriológico, mas o tempo requerido para o processamento das amostras e o diagnóstico final é longo, por isso existe a necessidade de desenvolvimento de métodos alternativos que visem acelerar esta etapa. Para isto utilizamos a separação imunomagnética associada ao bacteriófago P22 como técnica de detecção rápida para os seguintes sorovares de Salmonella: Salmonella enterica subsp. enterica sorovar Heidelberg (S. Heidelberg), Salmonella enterica subsp. enterica sorovar Enteritidis (S. Enteritidis) e Salmonella enterica subsp. enterica sorovar Typhimurium (S. Typhimurium), os quais foram inoculados artificialmente em lavados de sobre-coxas de frango nas seguintes concentrações: 5, 10 e 100 UFC/25mL. A eficiência da técnica, representada pelo tempo requerido para detecção de amostras positivas ou negativas, foi comparado com os testes rotineiramente utilizados para detecção de Salmonella, o exame bacteriológico e a reação em cadeia da polimerase (PCR). Este estudo confirmou a capacidade do teste de separação imunomagnética associado a bacteriófago, o qual identificou 99,6% das amostras positivas para Salmonella, dos três sorovares testados. Já o bacteriológico e PCR identificaram respectivamente 95,1% e 98,5% das amostras positivas.(AU)

Detection of N-ethyl-N-nitrosourea-induced Pig-a gene mutation in rats by immunomagnetic separation technique combined with flow cytometry / 中国实验动物学报

Objective To optimize the detection test of Pig-a gene mutation in peripheral blood of rats by enriching and detecting mutant erythrocytes, using immunomagnetic separation technique in combination with flow cytometry. Methods SD rats were administered with 20,40 and 80 mg/(kg·bw)doses of N-ethyl-N-nitrosourea(ENU) continually for 3 days. The peripheral blood samples of rats were collected on the 7th,14th and 28th days, respectively, after treatment. Immunomagnetic separation columns were used to enrich RETCD59- and RBCCD59- cells, and then flow cytometry was used to count the number of pre-column and post-column peripheral erythrocytes. Results Compared with the control group,the frequencies of RETCD59- and RBCCD59- were significantly increased in each ENU group(P<0.05). With immunomagnetic separation technique, the test of Pig-a gene mutation of a sample could be completed within 3 minutes,and the number of detected RETCD59- or RBCCD59-cells was up to 2×104or 9×104, respectively. Conclusions In this study,immunomagnetic separation in combination with flow cytometry is used to establish and optimize the Pig-a gene mutation test in rat peripheral blood,showing a high-throughput detection and improved accuracy and efficiency of the experiment.

Synthesis and Characterization of Immunomagnetic Nanoparticles Modified by Monoclonal Antibodies Against Human Breast Mammaglobin / 中国药学杂志

OBJECTIVE: To synthesize immunomagnetic nanoparticles with uniform particle size, strong superamagnetism as well as strong immune activity which can be specifically and sensitively combined with circulating tumor cells in peripheral blood of patients with breast cancer.METHODS: Superparamagnetic oxide iron nanoparticles containing active carboxyl groups (SMNP-COOH) were synthesized by polyol methods, thermogravimetric analysis was used to determine the amount of carboxyl groups on the surface of SMNP-COOH, while the content of iron was determined by o-phenanthroline. Mediated by 1-ethyl-3,3-dimethylaminopropyl carbodiimide(EDC) and N-hydroxysuccinimide (NHS), immunomagnetic nanoparticles(IMNP) against human breast carcinoma cell line were constructed by binding the monoclonal antibodies against hMAM with SMNP-COOH. X-Ray diffraction was used to confirm their synthesis,meanwhile,transmission electron microscope (TEM), dynamic light scattering (DLS), and vibrating sample magnetometry (VSM) were applied to characterize their physicochemical properties. The conjugation amount of the antibodies and the activity of IMNPs were evaluated by enzyme linked immunosorbent assay (ELISA).RESULTS: X-Ray diffraction showed that the chracteristic peaks of the crystalline powder of SMNP-COOH and IMNP agreed with the Fe3O4 standard. The concentration of iron in SMMP-COOH and IMNP were 0.205 and 0.164 mol•L-1, respectively.TEM showed that both synthesized SMNP-COOH and IMNP were almost spherical or ellipsoidal. The sizes of SMNP-COOH and IMNP were (13.7±3.6) and (15.4±4.5) nm, respectively. Dynamic light scattering(DLS) demonstrated the intensity particle size and polydispersity index (PDI) of SMNP-COOH and IMNP were 23.4 nm and 0.303, and 71.2 nm and 0.175,respectively. VSM results showed that both SMNP-COOH and IMNP had strong superamagnetism, and the saturation magnetization of SMNP-COOH and IMNP were 71.37 and 67.68 emu•g-1Fe, respectively, which confirmed antibody binding may reduce the magnetism of SMNP-COOH. The ELISA results showed the conjugation amount of antibody was about 93 μg on 1 mg SMNP-COOH by covalent bond. The obtained immunomagnetic nanoparticles (IMNP) which were bound with the hMAM monoclonal antibodies could specifically and sensitively combine with breast cancer cell line MDA-MB-415.CONCLUSION: IMNP with strong superparamagnetic property,excellent stability and perfect antibody activity were successfully synthesized, which demonstrate the potential to magnetically separate circulating tumor cells in peripheral blood from patients with breast cancer, thus providing a favorable weapon to accurately detect CTCs in breast tumor patients.

Immunomagnetic Nanosphere-based Strategy for Rapid, Efficient and Accurate Detection of Lymphnode Metastatic Carcinoma Cells / 分析化学

A strategy based on immunomagnetic nanospheres ( IMNs ) for rapid, efficient and accurate detection of lymphnode metastasis carcinoma cells ( LNMCCs ) was developed in this study. First, IMNs processing magnetism and biological targeting were fabricated by the approach developed by our group previously. Then, LNMCCs in lymph node fine needle aspiration ( LNFNA) specimens were separated and enriched by the immunomagnetic isolation using IMNs. At last, the captured cells were identified with Wright's staining and immunocytochemistry ( ICC) . The separation and enrichment of LNMCCs with immunomagnetic isolation could reduce the background interference of LNFNA specimens effectively; the identification with Wright ' s staining and ICC offered more reliable information for accurate diagnosis, so the sensitivity, specificity and overall diagnostic accuracy had an obvious improvement compared with the conventional cytologic diagnosis. Besides, the simple and rapid incubation of LNFNA specimens with IMNs needed just 5 min, so the cytomorphology of captured LNMCCs could be intactly retained, which enabled to provide important basis for classifying lymphnode metastasis carcinoma ( LNMC ) and the subsequent pathological study. Moreover, the specific capture of epithelial carcinoma cells in LNFNA specimens with IMNs could make a definite diagnosis of the captured cells as LNMCCs, thus realizing the differentiated diagnosis of LNMC and malignant lymphoma. Additionally, this strategy exhibited successful LNMCCs detection in LNFNA specimens from 110 patients and had higher sensitivity ( 98 . 0%) , specificity ( 100 . 0%) , and overall diagnostic accuracy (98. 2%) than the conventional cytologic diagnosis. Therefore, it was a new attempt to use IMNs for detection of LNMCCs in LNFNA specimens from LNMC patients, and offered new ideas for LNMC diagnosis and study.

Fluorescent Microspheres Lateral Flow Assay Based on Immunomagnetic Separation for Detection of S.choleraesuis / 分析化学

Immunomagnetic separation (IMS) was coupled with fluorescent microspheres lateral flow assay (FM-LFA) for rapid detection of S.choleraesuis in this study.The target bacteria were firstly enriched from sample by immunomagnetic beads (IMBs),then eluted by heat treatment and detected by fluorescent microspheres lateral flow test strip.The IMBs was labeled with 30 μg/mg antibody,and the capture efficiency was greater than 90％ against 102-106 CFU/mL of S.choleraesuis with great specificity.The immunofluorescent microspheres were prepared by coupling 300 μg of 11 D8-D4 monoclonal antibody with 1 mg of fluorescent microspheres at pH 6.Monoclonal antibody 5F11-B11 (2.0 mg/mL) and donkey anti-mouse IgG (1.0 mg/mL) were sprayed on nitrocellulose membrane as test line and control line,respectively.The FM-LFA based on IMS was used to detect S.choleraesuis in PBS and milk.The limits of detection in PBS buffer and milk were 1.5×105 CFU/mL and 7.6×105 CFU/mL respectively,which were 10 and 200 times lower than that of traditional fluorescent microspheres lateral flow assay,respectively.The results showed that the method,which could enrich S.choleraesuis in milk effectively,could avoid matrix interference and improve the detection sensitivity,thus had a good application prospect.

Enhanced effect of immunomagnetic beads on micro-CT scan of the lung adenocarci-noma mouse model / 中国肿瘤临床

Objective:To study the signal enhancement of lung adenocarcinoma nude mice after injection of immunomagnetic bead solution (magnetic beads conjugated with monoclonal antibody NJ001) in micro-CT scan. Methods:The models of lung adenocarcino-ma nude mice were established by injecting SPC-A1-luc cells through the tail vein and were validated by bioluminescence imaging (BLI). The nude mice were divided into three groups: physiological saline group, bare magnetic bead group, and immunomagnetic bead group. Three groups of nude mice were injected with physiological saline, 750 nm bare magnetic bead solution, and immuno-magnetic bead solution via the tail vein every week, and micro-CT scan was taken before and 4 h after injection. Immunohistochemis-try (IHC) was used to detect the expression of antigen SP70 in tumor tissues. Results:The tumor was detected in the immunomagnetic bead group at the fourth week, whereas in the physiological saline and bare magnetic bead groups, the tumor was undetectable until the sixth week. The tumor intensities detected at the sixth week by micro-CT scan in the physiological saline, bare magnetic bead, and immunomagnetic bead groups were 59.05 ± 0.66, 60.69 ± 0.55, and 58.25 ± 0.32 before injection and 60.30 ± 1.83, 61.05 ± 0.68, and 67.41±3.82 after injection, respectively. Compared with the tumor intensities before injection, they significantly increased after injec-tion in the immunomagnetic bead group;the difference was statistically significant (P=0.0079). By contrast, no statistical significance was observed in the tumor intensities before and after injection in the physiological saline and bare magnetic bead groups (P=0.1867 and P=0.3839, respectively). Conclusion:The immunomagnetic beads had enhanced effect on micro-CT scan of lung adenocarcinoma nude mouse models.

Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR

Abstract The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900 -PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 101 CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina.

Método imunomagnético associado ao meio MesenCult® na obtenção de células mononucleares da medula óssea de coelhos negativas para o anticorpo monoclonal CD45 / Immunomagnetic method associated with MesenCult® medium to obtain bone marrow mononuclear cells from rabbits negative for CD45 monoclonal antibody

O objetivo detse artigo é de descrever um protocolo de isolamento das células mononucleares da medula óssea de coelhos, seguido de purificação celular por depleção negativa com o anticorpo monoclonal CD45 e posterior expansão em meio de cultura MesenCult®. Dez coelhos machos adultos, da raça Nova Zelândia, com idade média de 1,0±0,2 anos e peso médio 3,5±0,24kg, foram utilizados para padronização da metodologia. O isolamento das células mononuclares da medula óssea foi realizado pelo gradiente de densidade Ficoll-paque® e a purificação e obtenção das células- pela depleção negativa com o anticorpo monoclonal CD45 em base imunomagnética. A população celular obtida foi expandida posteriormente em meio de cultura MesenCult®. No isolamento pelo gradiente de icoll-Paque® foi obtido um rendimento médio de 7,31x106 células/mL. Após purificação e obtenção das possíveis células-tronco mesenquimais pela base imunomagnética, houve um decréscimo do rendimento para 2,28x106 células/mL, mas o processo de expansão foi incrementado pelo cultivo celular. Os resultados indicaram que as células obtidas da fração mononuclear da medula óssea, cultivadas in vitro foram capazes de gerar células aderentes 24 horas após o cultivo, com predominância de células fibroblastóides sugestivas de células-tronco mesenquimais. Concluiu-se que a obtenção de células-tronco mesenquimais pode ser alcançada após purificação das células mononucleares da medula óssea de coelhos pelo método imunomagético, o meio de cultura MesenCult® proporciona um ambiente adequado para a rápida expansão in vitro e o número de passagens exerce influência negativa sobre as características morfológicas das células.

The objective of this study was to describe guidelines for the isolation of bone marrow mononuclear cells from rabbits, followed by cell purification by negative depletion with CD45 monoclonal antibody, and further expansion in MesenCult® medium. Ten adult male New Zealand White rabbits, age average of 1.0±0.2 years and weighting 3.5±0.24kg, were used to obtain a standardized method. The mononuclear cells of the bone marrow were isolated with Ficoll-paque® density gradient centrifugation, and the cell purification and acquisition was completed by negative depletion with CD45 monoclonal antibody in immunomagnetic base. The cell population obtained was expanded in MesenCult® medium. Through isolation with Ficoll-paque® density gradient was possible to obtain an average yield of 7.31x106 cells/mL. After purification and acquisiton of potential mesenchymal stem cells by the immunomagnetic base, there was a yield decrease to 2.28x106 cells/mL; however the expansion process was increased in cell culture. The results indicated that cells obtained from the mononuclear fraction of bone marrow and cultivated in vitro were capable to generate adherent cells 24 hours after culture, with predominance of fibroblastoid cells suggestive of mesenchymal stem cells. It can be concluded that mesenchymal stem cells can be achieved with purified rabbit bone marrow mononuclear cells through the immunomagnetic method, as the MesenCult® medium provides a suitable environment for a quick in vitro expansion, and the number of passages exerts negative influence on the morphological characteristics.

Research progress on enrichment and isolation of circulating tumor cells using immunomagnetic separation based microfluidic technology / 中华检验医学杂志

Circulating tumor cells ( CTCs ) exists in peripheral blood of patients with a variety of malignant tumors , the accurate detection of CTCs helps early detection of cancer , and can be used to guide individualized treatment of cancer patients , rapid assessment of tumor chemotherapy drugs and detection of tumor recurrence .However , the CTCs in peripheral blood of cancer patients were extremely low , their detection requires efficient and specific enrichment and separation methods .The new microfluidic technology can accurately control the microliquid preciously , combined with the immunomagnetic separation technology , has been applied in detecting CTCs , which can save the blood samples of CTCs , has the advantages of simple operation , fast sorting speed , high specificity and high purity .

Study on quantitatively detecting Mycobacterium tuberculosis ESAT-6 antigen by using near-infrared fluorescent dye-immunomagnetic beads coupling method / 国际检验医学杂志

Objective To establish a near‐infrared fluorescent dye‐immunomagnetic beads coupling method for quantitative de‐tection of Mycobacterium tuberculosis early secretory antigenic target‐6(ESAT‐6) .Methods Near‐infrared fluorescent dye ,dylight 800 ,was used to mark ESTA‐6‐targeting monoclonal antibodies ,and the surface of nano‐magnetic beads were coated with ESAT‐6‐targeting polyclonal antibodies .Double antibody sandwich method was used for magnetic separation of conjugates and dissociants . Portable high‐sensitivity and low‐noise excitation fluorescence detector was used to detect the intensity of magnetic combination ,so as to test the ESAT‐6 content in test samples .Results The detecting linear range of this method was 2 .4-750 .0 ng/mL ,and the minimum detection limit was 0 .48 ng/mL .The recovery rate was 96% at a concentration of 10 ng/mL ,and at a concentration of 50 ng/mL the recovery rate was 95% .The between‐run coefficient of variation(CV)value was 5 .8% ,and the within‐run CV value was 4 .3% .The specificity and sensitivity of this method for detecting ESAT‐6 in clinical pleural effusion samples were 80% and 95% , respectively .Conclusion This method might have wide linear range ,high sensitivity and good stability in the detection of ESAT‐6 .

Integrating immunomagnetic bead enrichment with immunochromatography for the detection of in-fluenza A virus / 中华微生物学和免疫学杂志

Objective To establish a novel method by integrating immunomagnetic bead enrich-ment with immunochromatography for the detection of influenza A virus. Methods The immunomagnetic beads were prepared by using EDC/NHS method and then coupled with monoclonal antibodies against influ-enza A virus. A direct immunomagnetic beads-based immunochromatography for the detection of influenza A virus was developed by using double-antibody sandwich method and immunochromatography, which was fur-ther combined with immunomagnetic separation to establish the novel integrated method of immunomagnectic bead enrichment and immunochromatography. Clinical throat swab samples collected from patients with influ-enza A virus infection and healthy subjects were analyzed by the novel method and the results were compared with those by using the conventional colloidal gold immunochromatography to evaluate the specificity, sensi-tivity and positive coincidence rate of this established method. Results The direct immunomagnetic beads-based immunochromatography and the colloidal gold immunochromatography showed no significant differences in specificity and sensitivity and could be used to identify influenza A virus-positive samples with cycle threshold ( Ct) values less than or equal to 22 obtained by real-time PCR assay. The integrated method could identify positive samples with Ct values less than or equal to 28, indicating that the novel method was more sensitive. Conclusion The novel method by integrating immunomagnetic bead enrichment with immunochroma-tography was successfully established and suitable for the rapid and on-site detection of influenza A virus.

Extraction of prostate cancer stem cells using self-designed magnetic beads / 中国组织工程研究

BACKGROUND:Effective sorting of prostate cancer stem cels is the basis of experimental studies in prostate cancer developing. The most common sorting method is magnetic-activated cel sorting. OBJECTIVE:To separate CD133+/CD44+ cels in prostate cancer tissues using self-designed magnetic beads folowed by culture, passage and immunological identification. METHODS:Self-designed magnetic microspheres were applied to establish immunomagnetic beads to sort CD133+/CD44+ cels in prostate cancer tissues. The sorted cels were cultured in serum-free medium. The sphere formation, cel morphology, and proliferation ability after cel passage were statisticaly compared between the sorted cels and the normal tumor cel lines. Immunofluorescence detection was performed to detect the expression of specific antibodies. RESULTS AND CONCLUSION:Self-designed immunomagnetic beads had smal diameter and a high-sorting effect. The sorted cels possessed a high capacity of microsphere formation. After cel culture and passage, the cels highly expressed CD133 and CD44 antigens. The sorted cels with no induction had varying shapes and grew vigorously. After induction with transforming growth factor-β, the cultured cels were noted to have a single shape and grow slowly. The cel proliferation ability of sorted cels in these two groups differed significantly from that of the normal cancer cel lines (bothP < 0.05). In conclusion, the CD133+/CD44+ cels sorted from prostate tumor cels possessed cel morphology and function characteristics of stem cels which can provide a basis for extraction and culture of prostate cancer stem cels. Cite this article:Gong R, Li SY, Huo ZX, Ding H, Sun EL. Extraction of prostate cancer stem cels using self-designed magnetic beads. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):36-41.

Progress in the isolation and purification methods of adipose-derived stem cells / 中国比较医学杂志

Adipose-derived stem cells ( ASCs ) as potential seeded cells have been widely used in tissue engineering.Thus to obtain enough, high activity, high purity adipose-derived stem cells is the particular important premise of the application in tissue engineering.In this paper, the isolation and purification methods of ASCs were reviewed and the merit and demerit of different methods were compared in order to provide theoretical basis for safe and high-effective isolation and purification of ASCs.