Affinity purification of mAb from serum-containing hybridoma culture supernatant through a novel nanobody that discriminates mouse IgG from bovine IgG by recognizing the mouse kappa constant region (mCK).

When purifying mAb from serum-containing hybridoma culture supernatant, it is essential that mouse IgG remains free from contaminations of bovine IgG. However, the broadly used Protein A resin cannot achieve this goal due to binding between both mouse and bovine IgG. Here, a novel nanobody-based affinity purification magnetic beads that discriminates mouse IgG from bovine IgG was developed. To bind all subtypes of mouse IgG (IgG1, IgG2a, IgG2b and IgG3) that contain the kappa light chain, mCK (mouse kappa constant region)-specific nanobody binders were selected from an immune phage display VHH library; this library was constructed with peripheral blood mononuclear cells (PBMCs), which were collected from Bactrian camels immunized with a mix of intact mouse IgGs (IgG1, IgG2a, IgG2b and IgG3). A novel clone that exhibited a higher expression level and a higher binding affinity was selected (4E6). Then, the 4E6 nanobody in the format of VHH-hFC (human Fc) was conjugated on magnetic beads with a maximal binding capacity of 15.41±0.69 mg mouse IgG/mL beads. Furthermore, no bovine IgG could be copurified from hybridoma culture supernatant with immunomagnetic beads. This approach is valuable for the large-scale in vitro production of highly pure antibodies by hybridoma cells.

Development of a broad-spectrum one-step immunoassay for detection of glucocorticoids in milk using magnetosome-based immunomagnetic beads.

Immunomagnetic beads provide novel tools for high-throughput immunoassay techniques. In this study, protein G (PG) was immobilized onto bacterial magentic particles (BMPs) using an additional cysteine residue at the C-terminus. A broad-spectrum monoclonal antibody against glucocorticoids (GCs) was attached to BMPs through PG-Fc interaction, generating BMP-PG-mIgG immunomagentic beads. A sensitive one-step immunoassay was developed for GCs based on combination of BMP-PG-mIgG and dexamethasone-horseradish peroxidase tracer (DMS-HRP). The developed assay exhibited half inhibitory concentrations (IC50) for dexamethasone (DMS), betamethasone (BMS), prednisolone (PNS), hydrocortisone (HCS), beclomethasone (BCMS), cortisone (CS), 6-α-methylprednisone (6-α-MPNS), fludrocortisone acetate (HFCS) of 0.98, 1.49, 2.42, 9.29, 1.63, 6.13, 7.3, and 4.89 ng/mL, respectively. The method showed recoveries ranging rates from 86.5 % to 117 % with a coefficient of variation less than 12.3 % in milk sample, which showed a good correlation with LC-MS/MS. Thus, the proposed assay offers a rapid and broad-spectrum screening tool for simultaneous detection of GCs in milk.

One-Step Purification of IgE Epitope-Specific Antibody Using Immunomagnetic Beads and Highly Sensitive Detection of Bovine ß-Lactoglobulin for the Prediction of Milk Allergenicity in Foods.

Bovine ß-lactoglobulin (BLG) is a common allergen found in milk, and the immunoglobulin E (IgE) epitope plays a crucial role in cow milk allergy. Therefore, targeting the IgE epitope could be useful in accurately detecting BLG and assessing its allergenicity. However, producing an IgE epitope-specific antibody (IgE-EsAb) through traditional methods requires complex and time-consuming procedures. Here, IgE-EsAb was purified from rabbit anti-BLG sera by immunomagnetic beads in one step. Then, a sandwich ELISA (sELISA) based on the IgE-EsAb was developed to detect BLG and predict the potential milk allergenicity in foods. The obtained IgE-EsAb could specifically recognize the target IgE epitope of BLG and exhibited high affinity and specificity. The developed IgE-EsAb-based sELISA demonstrated an ultra-wide linear range of 3.9-1.28 × 105 ng/mL, with a limit of detection of 0.49 ng/mL for BLG. Additionally, the proposed immunoassay showed high specificity and recoveries (91.24-109.61%). The ability of the IgE-EsAb-based sELISA to evaluate the potential milk allergenicity in foods was validated using sera from cow milk allergy patients. These results suggest that immunomagnetic beads are an effective tool for rapidly obtaining the IgE-EsAb, and our proposed sELISA could be a reliable and user-friendly method for monitoring trace amounts of BLG and predicting the potential milk allergenicity of food samples.

Automatic Pretreatment of Dispersive Liquid Liquid Microextraction Based on Immunomagnetic Beads Coupled with UPLC-FLD for the Determination of Zearalenone in Corn Oils.

Sample pretreatment is a vital step in the detection of mycotoxins, and traditional pretreatment methods are time-consuming, labor-intensive and generate much organic waste liquid. In this work, an automatic, high-throughput and environmentally friendly pretreatment method is proposed. Immunomagnetic beads technology and dispersive liquid-liquid microextraction technology are combined, and the zearalenone in corn oils is directly purified and concentrated under the solubilization effects of surfactant. The proposed pretreatment method allows for the batch pretreatment of samples without pre-extraction using organic reagents, and almost no organic waste liquid is produced. Coupled with UPLC-FLD, an effective and accurate quantitative detection method for zearalenone is established. The recovery of spiked zearalenone in corn oils at different concentrations ranges from 85.7 to 89.0%, and the relative standard deviation is below 2.9%. The proposed pretreatment method overcomes the shortcomings of traditional pretreatment methods and has broad application prospects.

Preparation of polyclonal antibody against a universal bacterial antigen OmpA deduced by bioinformatic analysis and preliminary evaluation of concentration effects on foodborne pathogens.

Rapid and ultrasensitive microbial detection in actual samples have challenges because of target pathogen diversity and low abundance. In this study, we attempted to capture and concentrate multiple pathogens by combining magnetic beads with polyclonal antibodies against a universal antigen of ompA, LAMOA-1, before further detection. A protein sequence consisting of 241 amino acids with spatial conformation similar to E. coli ompA was identified and expressed as a recombinant protein in prokaryotes according to the results of sequence alignment among 432 sequences of ompA belonging to intestinal bacteria from gram-negative bacteria. Purified from immunized rabbits, the anti-LAMOA-1 antibody was shown to effectively recognize 12 foodborne bacterial species. Antibody-conjugated beads were used to concentrate the bacteria when the bacterial concentration in artificially contaminated samples is between 10 and 100 CFU/mL, which shortens detection duration by 8-24 h. The enrichment strategy is potentially beneficial for detection of foodborne pathogens.

Isolation of Fusobacterium nucleatum from human feces using immunomagnetic separation coupled with fastidious anaerobe agar.

AIM: Fusobacterium nucleatum (F. nucleatum) is associated with the initiation, development, and metastasis of colorectal cancer. However, it is difficult to isolate F. nucleatum from clinical specimens. In this study, we aimed to develop an effective and rapid method for isolating F. nucleatum from human feces using polyclonal antibody (PAB)-coated immunomagnetic beads (IMBs) with selective media. METHODS AND RESULTS: IMBs conjugated with PAB were prepared and used to isolate F. nucleatum from human feces, and the bacteria were cultured with selective culture media (fastidious anaerobe agar + nalidixic acid + vancomycin). Under optimized experimental conditions, IMBs could selectively recover F. nucleatum from fecal microbiota samples spiked with Peptostreptococcus or Bacteroides fragilis. In artificial fecal samples, the detection sensitivity of IMBs for F. nucleatum was 103 CFU mL-1. In addition, IMBs combined with selective media could rapidly isolate F. nucleatum from human feces. CONCLUSIONS: This study successfully established an effective method for the rapid isolation of F. nucleatum from human feces by IMBs. The whole procedure requires 2-3 days, and has a sensitivity of 103 CFU mL-1 feces.

Rapid Detection of Staphylococcus aureus in Milk and Pork via Immunomagnetic Separation and Recombinase Polymerase Amplification.

Separation processes using immunomagnetic beads (IMBs) are advantageous for the rapid detection of Staphylococcus aureus (S. aureus). Herein, a novel method, based on immunomagnetic separation using IMBs and recombinase polymerase amplification (RPA), was employed to detect S. aureus strains in milk and pork. IMBs were formed by the carbon diimide method using rabbit anti-S. aureus polyclonal antibodies and superparamagnetic carboxyl-Fe3O4 MBs. The average capture efficiency for 2.5 to 2.5 × 105 (CFU)/mL gradient dilution of S. aureus with 6 mg of IMBs within 60 min were a range of 62.74 to 92.75%. The detection sensitivity of the IMBs-RPA method in artificially contaminated samples was 2.5 × 101 CFU/mL. The entire detection process was completed within 2.5 h, including bacteria capture, DNA extraction, amplification, and electrophoresis. Among 20 actual samples, one case of raw milk sample and two cases of pork samples were tested positive using the established IMBs-RPA method, which were verified by the standard S. aureus inspection procedure. Therefore, the novel method shows potential for food safety supervision owing to its short detection time, higher sensitivity, and high specificity. IMPORTANCE Our study established IMBs-RPA method, which simplified the steps of bacteria separation, shortened the detection time, and realized the convenient detection of S. aureus in milk and pork samples. IMBs-RPA method was also suitable for the detection of other pathogens, providing a new method for food safety monitoring and a favorable basis for rapid and early diagnosis of diseases.

Isolation of atrazine nanobodies enhanced by depletion of anti-carrier protein phages and performance comparison between the nanobody and monoclonal antibody derived from the same immunogen.

Nanobody, a single domain antibody, has been shown a great promise for immunoassay (IA) applications. To improve the panning efficiency so as to obtain a valuable nanobody, anti-carrier protein phages in a phage display library were depleted to enhance the selection of nanobodies against the herbicide atrazine by using immunomagnetic beads conjugated with bovine serum albumin (IMB-BSA). The depletion of anti-carrier protein phages from the atrazine phage display library tripled the number of atrazine positive phage clones after four rounds of panning. One of the most sensitive phage clones Nb3 selected from the IMB-BSA depleted library was used to compare the performance with the monoclonal antibody (mAb 5D9) developed from the same immunogen. The Nb3-based IA exhibited similar specificity with the mAb 5D9-based IA, but greater thermostability and organic solvent tolerance. The half-maximum inhibition concentration (IC50) of the former was 3.5-fold greater than that of the latter (36.7 ng/mL versus 10.2 ng/mL). Because the Nb3-based IA was more robust than the mAb 5D9-based IA, the method detection limit of the two assays was 7.8 ng/mL of atrazine in river samples. The depletion strategy can increase the chance to acquire high quality nanobody and can be applicable for effective development of nanobodies against other small molecules.

Method of transfection of STOP gene lentiviral vector into oligodendrocyte precursor cells of BTBR mouse model of autism / 解剖学报

Objective To investigate the effect of microtubule binding protein STOP on myelin formation of oligodendrocyte in BTBR mice spectrum disorder in vitro, a highly purified primary culture method of oligodendrocyte precursor cells from cerebral cortex of BTBR mice was established. Establishment of a highly efficient transfection method for overexpression of STOP gene in oligodendrocyte precursor cells of BTBR mice cerebral cortex using lentiviral vector. Methods BTBR mice were used as experimental objects, 6-10 suckling mice were taken each time, repeat 3 times independently. The single cell suspension was prepared by trypsin digestion, and the primary oligodendrocyte precursor cells were obtained by immunomagnetic bead cell sorting method . After 5 days of culture, the cell purity was identified by oligodendrocyte precursor cell marker staining. The primary cultured oligodendrocyte precursor cells were transfected with STOP gene vector constructed in the early stage of the project group. 72-96 hours after transfection, the fluorescence staining of oligodendrocyte precursor cells was observed under fluorescence microscope, and the transfection rate and cell survival rate were calculated. Results The oligodendrocyte precursor cells of BTBR mice extracted by immunomagnetic beads sorting method basically adhered to the wall completely after 48 hours, and the cells had strong ability of proliferation. On the fifth da)' of culture, the purity of the cells was more than 95% identified by immunofluorescence. A lentivirus transfection method for primary oligodendrocyte precursor cells of BTBR mice with high transfection efficiency was established. The fluorescence expression of the cells was obvious after being photographed by high connotation microscope, the lentivirus transfection rate of oligodendrocyte precursor cells was increased to 60%-70%. Conclusion The primaiy oligodendrocyte precursor cells of BTBR mouse cerebral cortex with high purity were successfull)' isolated and cultured. A method for lentivirus infection of primaiy oligodendrocyte precursor cells in the cerebral cortex of BTBR mice is successfully established.

pH-Regulated Strategy and Mechanism of Antibody Orientation on Magnetic Beads for Improving Capture Performance of Staphylococcus Species.

Immunomagnetic beads (IMBs) have been widely used to capture and isolate target pathogens from complex food samples. The orientation of the antibody immobilized on the surface of magnetic beads (MBs) is closely related to the effective recognition with an antigen. We put forward an available strategy to orient the antibody on the surface of MBs by changing the charged amino group ratio of the reactive amino groups at optimal pH value. Quantum dots labeling antigen assay, antigen-binding fragment (Fab) accessibility assay and lysine mimicking were used for the first time to skillfully illustrate the antibody orientation mechanism. This revealed that the positively charged ε-NH2 group of lysine on the Fc relative to the uncharged amino terminus on Fab was preferentially adsorbed on the surface of MBs with a negatively charged group at pH 8.0, resulting in antigen binding sites of antibody fully exposed. This study contributes to the understanding of the antibody orientation on the surface of MBs and the potential application of IMBs in the separation and detection of pathogenic bacteria in food samples.

Simple Staining of Cells on a Chip.

Simple staining of cells is a widely used method in basic medical diagnostics, education, and research laboratories. The stains are low-cost, but the extensive consumption results in excessive toxic waste generation. Thus, to decrease the amount of toxic waste resulting from the cell staining procedure is a need. In this study, we developed a magnetically driven and compartmentalized passive microfluidic chip to perform simple staining of human eukaryotic cells, K562 cells, and lymphocyte cells derived from patients. We demonstrated simple staining on cells with trypan blue, methylene blue, crystal violet, and safranin for high, medium, and low cell densities. The stained cells were imaged using a bright field optical microscope and a cell phone to count cells on the focal plane. The staining improved the color signal of the cell by 25-135-pixel intensity changes for the microscopic images. The validity of the protocol was determined using Jurkat and MDA-MB-231 cell lines as negative controls. In order to demonstrate the practicality of the system, lymphocyte cells derived from human blood samples were stained with trypan blue. The color intensity changes in the first and last compartments were analyzed to evaluate the performance of the chip. The developed method is ultra-low cost, significantly reduces the waste generated, and can be integrated with mobile imaging devices in terms of portability. By combining microfabrication technology with cell staining, this study reported a novel contribution to the field of microfluidic biosensors. In the future, we expect to demonstrate the detection of pathogens using this method.

Monovalent Antigen-Induced Aggregation (MAA) Biosensors Using Immunomagnetic Beads in Both Sample Separation and Signal Generation for Label-Free Detection of Enrofloxacin.

Exploring new functions of nanomaterials can help facilitate the development of biosensors for the detection of antibiotics. Herein, a new detection modality based on monovalent antigen-induced aggregation (MAA) of immunomagnetic beads (IMBs) was proposed for rapid and label-free detection of enrofloxacin (ENR), which endowed IMBs with the abilities of both sample separation and signal generation. In the presence of ENR, the initially well-dispersed IMBs were aggregated and the degree of aggregation was in a concentration-dependent manner. After exploring the mechanism underlying IMB aggregation and investigating the key parameters affecting it, a label-free biosensing platform was developed for rapid and sensitive detection of ENR. Based on the significant differences in the magnetic separation speed and size between the aggregated and well-dispersed IMBs, two methods were proposed for quantitatively determining ENR, i.e., measuring the turbidity of the IMB supernatant after magnetic separation for a given time and visualizing and calculating the grayscale value of the aggregated IMBs trapped on the surface of a nitrocellulose membrane. A three-dimensional (3D)-printed syringe was designed and fabricated for automatic filtration of IMBs. This immunosensor allowed for sensitive detection of ENR in less than 15 min without any labels. It exhibited a satisfactory limit of detection of 0.79 ng mL-1 and showed the feasibility for ENR detection of spiked chicken meat with recovery rates ranging from 74.8 to 98.3%. The MAA immunosensor can act as a promising tool to detect trace levels of ENR and has the potential to be applied to complex food samples.

Rapid Detection of Single Viable Escherichia coli O157 Cells in Fresh Lettuce and Strawberry by Immunomagnetic Flow Cytometry in Combination with Pre-Enrichment.

Enterohemorrhagic Escherichia coli are an important pathogen causing food poisoning. The rapid detection of viable E. coli O157 in vegetables and fruits at single-cell level is critical because of the low infective dose of this pathogen. In this study, an immunomagnetic flow cytometry (IMFC)-based method was developed to detect E. coli O157 in lettuce and strawberries inoculated with 1 CFU/25 g. This method developed immunomagnetic (IM)-beads to capture E. coli O157 cells. The pre-enrichment of E. coli O157 and IM-bead separation rapidly increased the concentration of cells to a detectable range for flow cytometry. Compared with the plate-based method, the diagnostic sensitivity and specificity of the IMFC-based method were 100% in 166 samples, including 100 artificially contaminated samples, 60 retail samples, and six O157-positive samples for proficiency testing. The developed IMFC-based method was found to be effective in detecting E. coli O157 at single-cell level in 25 g of lettuce or strawberry with relatively shorter associated time to results of 5.7 h. Therefore, the IMFC-based method could improve detection efficiency and also make early warnings in a short time.

Development of a time-resolved fluorescence immunoassay based on immunomagnetic beads for gastrin-17.

OBJECTIVE: In this study, a novel, simple, and rapid immunoassay for the determination of gastrin-17 (G-17) in human serum was established by combining immunomagnetic beads with time-resolved fluorescence immunoassay (TRFIA). METHODS: Immunomagnetic beads were coated with anti-G-17 M01 antibody, anti-G-17 M02 antibody was labeled with Eu3+ chelates. The concentration of G-17 in the serum was detected with the double-antibody sandwich method. RESULTS: The limit of background(LOB), limit of detection (LOD), and limit of quantification (LOQ) were 0.09, 0.104, and 0.39 pmol/L, respectively. The detection range of G-17-TRFIA was 0.39-100 pmol/L. The average intra- and inter-assay coefficients of variation (CV) were 5.95%-9.07% and 6.09%-8.14%, respectively. The recoveries for the serum samples ranged from 94.70% to 100.95%. The specificity of our G-17-TRFIA was acceptable. The correlation coefficient between G-17-TRFIA and commercial G-17-ELISA methods was R2 = 0.9092. CONCLUSIONS: A novel G-17-TRFIA detection method was successfully established to provide a reference for the early diagnosis of patients with atrophic gastritis in clinical research.

Improved isolation and detection of toxigenic Vibrio parahaemolyticus from coastal water in Saudi Arabia using immunomagnetic enrichment.

BACKGROUND: Vibrio parahaemolyticus is recognized globally as a cause of foodborne gastroenteritis and its widely disseminated in marine and coastal environment throughout the world. The main aim of this study was conducted to investigate the presence of toxigenic V. parahaemolyticus in costal water in the Eastern Province of Saudi Arabia by using immunomagnetic separation (IMS) in combination with chromogenic Vibrio agar medium and PCR targeting toxR gene of species level and virulence genes. METHODS: A total of 192 seawater samples were collected from five locations and enriched in alkaline peptone water (APW) broth. One-milliliter portion from enriched samples in APW were mixed with an immunomagnetic beads (IMB) coated with specific antibodies against V. parahaemolyticus polyvalent K antisera and separated beads with captured bacteria streaked on thiosulfate citrate bile salts sucrose (TCBS) agar and CHROMagar Vibrio (CaV) medium. RESULTS: Of the 192 examined seawater samples, 38 (19.8%) and 44 (22.9%) were positive for V. parahaemolyticus, producing green and mauve colonies on TCBS agar and CaV medium, respectively. Among 120 isolates of V. parahaemolyticus isolated in this study, 3 (2.5%) and 26 (21.7%) isolates of V. parahaemolyticus isolated without and with IMB treatment tested positive for the toxin regulatory (toxR) gene, respectively. Screening of the confirmed toxR gene-positive isolates revealed that 21 (17.5%) and 3 (2.5%) were positive for the thermostable direct hemolysin (tdh) encoding gene in strains isolated with IMB and without IMB treatment, respectively. None of the V. parahaemolyticus strains tested positive for the thermostable related hemolysin (trh) gene. In this study, we found that the CaV medium has no advantage over TCBS agar if IMB concentration treatment is used during secondary enrichment steps of environmental samples. The enterobacterial repetitive intergenic consensus (ERIC)-PCR DNA fingerprinting analysis revealed high genomic diversity, and 18 strains of V. parahaemolyticus were grouped and identified into four identical ERIC clonal group patterns. CONCLUSIONS: The presented study reports the first detection of tdh producing V. parahaemolyticus in coastal water in the Eastern Province of Saudi Arabia.

Isolation and Characterization Methods of Human Invariant NKT Cells.

Natural killer T cells (NKT) are abundant in the hepatic sinuses and account for about 20-50% of rat liver lymphocytes. Type I or invariant NKT cells (iNKT) exert a powerful pro-inflammatory effect when activated, while type II NKT cells are more heterogeneous and mainly play an immunomodulatory role. Here we mainly introduced the isolation and characterization methods of human invariant NKT cells. Through immunomagnetic beads and flow cytometry, iNKT cells can be isolated specifically, and that explains functional analysis can be further established.

Inertial-Assisted Immunomagnetic Bioplatform towards Efficient Enrichment of Circulating Tumor Cells.

Serving as an effective biomarker in liquid biopsy, circulating tumor cells (CTCs) can provide an accessible source for cancer biology study. For the in-depth evaluation of CTCs in cancer analysis, their efficient enrichment is essential, owing to their low abundance in peripheral blood. In this paper, self-assembled immunomagnetic beads were developed to isolate CTCs from the ordered bundles of cells under the assistance of the spiral inertial effect. Parametric numerical simulations were performed to explore the velocity distribution in the cross section. Based on this chip, rare CTCs could be recovered under the throughput of 500 µL/min, making this device a valuable supplement in cancer analysis, diagnostics, and therapeutics.

Development of IMBs-qPCR detection method for Yersinia enterocolitica based on the foxA gene.

Yersinia enterocolitica is an important zoonotic pathogen, which seriously endangers food-safety risk. In this study, the recombinant outer membrane protein OmpF and its antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Y. enterocolitica in food samples, combining the quantitative PCR detection with primers of virulence factor gene foxA for Yersinia enterocolitica contamination. The results showed that the capture efficiency of approximately 80% using anti-OmpF antibody-immunomagnetic beads and linearly dependent capture under 101-105 CFU/mL Y. enterocolitica compared with less than 10% capture of other bacteria. The detection limit of 64 CFU/mL was obtained based on foxA gene PCR detection combined with capture of the anti-OmpF antibody-immunomagnetic beads to detect Yersinia enterocolitica in artificially contaminated milk and pork samples. Compared to the culture method, the developed IMBs-qPCR method has higher consistency, was less time consuming, which taken together provides an effective alternative method for rapid detection of Y. enterocolitica in food.

Preparation of immunomagnetic beads coupled with a rhodamine hydrazine immunosensor for the detection of Mycobacterium avium subspecies paratuberculosis in bovine feces, milk, and colostrum.

The aim of this study was to develop and evaluate a method for detecting Mycobacterium avium ssp. paratuberculosis (MAP) bacteria in bovine fecal, milk, and colostrum samples using immunomagnetic beads (IMB) and a rhodamine hydrazone immunosensor. Immunomagnetic beads were prepared by using purified antibodies from hyperimmunized sera that were coupled to Fe nanoparticles with diethylene triamine pentaacetic acid (DTPA) or ethyl (dimethyl aminopropyl) carbodiimide (EDC)-N-hydroxy succinimide (NHS) as linkers. Rhodamine hydrazone particles were synthesized and coupled to IgY anti-MAP antibodies using DTPA or EDC-NHS linkers. Separation efficiency of the IMB was tested on bovine fecal, milk, and colostrum samples experimentally contaminated with MAP. The studied methods were evaluated on their ability to detect MAP and separate bacteria in complex mediums. The ELISA results indicated 95% efficacy in antibody coupling to IMB, with the DTPA-IMB method being more efficient than the EDC-NHS-IMB method. By using the DTPA-IMB method, MAP bacteria were successfully recovered from fecal, milk, and colostrum samples. The DTPA-IMB method used in combination with the rhodamine hydrazone immunosensor had a limit of detection equal to 30 and 30,000 MAP cells/mL using chromogenic and fluorescent properties, respectively. Combining the DTPA-IMB separation method with the rhodamine hydrazone immunosensor provides a fast, sensitive, and cost-beneficial method for detecting MAP in bovine feces, milk, and colostrum.

Multi-Marker Immunomagnetic Enrichment of Circulating Melanoma Cells.

Within the last decade, circulating tumor cells (CTCs) have emerged as a promising biomarker for prognostication, treatment monitoring, and detection of markers of treatment resistance, and their isolation can be used as a minimally invasive means of profiling tumors across multiple body sites. However, CTCs represent a minuscule fraction of the total circulating cells in a patient. Therefore, sensitive isolation methods are needed for the detection and downstream analysis of these cells. Herein we describe a sensitive method for melanoma CTC isolation using a multi-marker immunomagnetic bead method. This method has been purposely optimized to detect CTCs in melanoma patients.