Related Resources & Cores
Mucosal Immunology Core
Flow Cytometry Core Laboratory
, Core Manager
The Immuno/BioSpot Core specializes in assays related to the detection, quantification, and qualification of biological spot assays and single-cell related analyses, including ELISPOT, colony counting, plaque assays, FluoroSpot, cell viability tests, apoptosis tests, in vivo/in vitro cytotoxicity measurements using cells labeled with fluorescent dyes, histochemistry stains, genotoxicity assays, multi-color intracellular/surface quantification, etc.
The Core's latest specialization adds systems for analysis of multiple proteins, peptides, and nucleic acids in a single well as suspended in a variety of solution types (blood, media, tears, urine, milk) with the addition of a Luminex-based multiplexing array and assay-related support facilities and equipment alongside regulatory compliant software and validation tools for reliable and high-throughput multiplex assay results.
- Immuno/BioSpot Services include:
- Operator assisted acquisition and analysis
- Operator unassisted analysis and acquisition (for investigators who have taken the instruction course)
- Post acquisition data analysis, study, quality control, and file assembly/formatting.
- ELISPOT / FluoroSpot assay preparation
- Multiplex assay preparation
- ELISPOT / FluoroSpot assay materials, reagents, and Ab pairs
- Multiplex assay materials, reagents, and equipment
- Multiplex assay facilities including magnetic bead plate washing, micro-plate shaker, and staging platforms and equipment for various assay types.
- Assistance in development of unique bead-based multiplex assays with multiplex COOH beads by coupling with antibodies, antigens, receptors, ligands, enzymes, or nucleic acids.
- Instrumentation/Software Instruction (permitting unassisted supervised usage of Analyzers).
- Blank media, disks, and photo printouts
- ELISPOT / FluoroSpot Assay instruction
- Multiplex assay instruction
- Free assay consultation
- Immuno/BioSpot Assay Analysis offered:
- Quantitative and qualitative ELISPOT assays for cytokines and protein secretions
- FluoroSpot assays.
- Fluorescence-based Cell Counting / FluorSpot nuclear counting (to count hyperplasia, tissue infiltration, or cell counting).
- Microbial assays
- Colony studies
- Genotoxic assays
- Viral plaque assays
- Stained cell counting from histochemistry slides and sections, including multiple color fluorescent slide images.
- Multiplex Assay Analysis offered:
- Enzyme assays
- Receptor-ligand assays
- Nucleic acid hybridization assays
- Multiplex Assays in a Variety of Panel Configurations for
- Cytokines, chemokines, and growth factors
- The Th17 pathway
- Cell signaling
- Acute phase
- Custom designed multiplex bead assays
- Protein Quantitation
- Gene Expression
- Genotyping (SNP)
- Ratio Metric (Qualitative)
- Assay Descriptions:
- ELISPOT assays for cytokines and protein production measured at single cell level permitting rapid analysis for exceptional high throughput and extraction of detailed information enumerated from large sample numbers.
- FluoroSpot assays, permitting directly labeled detection, no enzymatic amplification or substrate, and six-color simultaneous detection permitting fewer cells and reagents.
- Fluorescence-based Cell Counting: powerful alternative to flow cytometry being: faster (with up to one million cells in an image analyzed in a couple seconds vs. much longer for the same number of cells by flow), more sensitive for rare cells (detection limit one fluorescent cell per million bystander cells i.e. 0.0001%), more economical (every cell is counted, none lost in system), lends itself to high-throughput versus flow, viability counts (live/dead/apoptotic cell counts), FluoroSpot nuclear counting (to count hyperplasia, tissue infiltration, or cells).
- Microbial assays: microbial load and bioburden testing--enabling colony miniaturization and automated analysis, increasing speed, throughput, and reproducibility.
- Colony studies permitting automated size-selection and enumeration for clonogenic assays and identifying multipotent progenitor cells.
- Genotoxic assays for imaging, measuring, and quantifying mutagenic potential by counting cells (mouse lymphoma assays) or microbes (e.g. Ames test).
- Viral plaque assays performing multiple object-oriented morphometric measurements, enabling user-defined gating by imaging and analyzing the monolayer or bacterial lawn.
- Quantification of immunohistochemistry and immunofluorescence slides and stains, including multiple-color fluorescent slide images--providing objective quantification along with a recorded image.
- Multiplex immunoassays analyzing broad selection of inflammation and immunology biomarkers available in a variety of human and animal models to investigate the modulation and expression of dozens of analytes simultaneously, with advantage of speed and sensitivity, which can improve productivity.
- Multiplex enzyme assays investigating cellular metabolites applicable to multiple research areas and diseases, including neurodegeneration, aging, metabolic syndrome, obesity, cancer, cardiovascular disease and toxicity.
- Multiplex receptor-ligand assays to obtain quantitative data: including absolute concentrations using synthetic peptides as standards; measurement of key signaling pathways with analytically validated, preconfigured kits; measurement multiple phosphorylation sites on the same protein in the same well, measure both total and phosphorylated protein in the same well.
- Multiplex nucleic acid hybridization assay by multiplexing of up to 100 custom-targets in one well using the suspension array system; simple single nucleotide polymorphism (SNP) genotyping using specialty software, specifically designed for integrated allelic discrimination; specialty software analysis for applications in SNP analysis, pathogen detection, strain typing, halotyping, and gene expression.
- Multiplex Assays in a variety of Panel Configurations for cytokines, chemokines, growth factors, TGF-b, the Th17 pathway, cell signaling, Cancer, Diabetes, Isotyping, Acute phase, custom designed multiplex bead assays.
- Multiplex Protein Quantitation to simultaneously examine multiple phosphorylation events involving upstream or downstream molecules in one or more cellular signaling pathways, maximizing information obtained from a single sample by permitting the analysis of more than one target protein at a time, with minimal sample volume requirements for evaluating the phosphorylation states of specific phosphoproteins or the expression levels of total target proteins.
- Multiplex Gene Expression to quantitatively measure multiple RNA targets simultaneously with high accuracy and precision, quantitation of original RNA population directly from lysates avoids biases, sample loss, false positive/negative results associated with RNA isolation, cDNA synthesis, PCR amplification. High accuracy and precision using branch DNA (bDNA) signal amplification versus target amplification for multiplexing 3 to 80 target RNAs and precision results in difficult clinical research samples, including, H&E-stained FFPE, blood, and skin.
- Multiplex Genotyping (SNP) - low to medium throughput genotyping projects, capable of analyzing anywhere from one SNP in one subject or sample or up to 50 SNPs in unlimited numbers of subject. The fluorescent microspheres used in the SNP assay are coupled to oligonucleotides (a "TAG sequence") such that each bead's fluorescent address is associated with a known nucleotide sequence. User-provided oligos, comprised of a SNP specific sequence coupled to the complement of the bead-associated oligo sequence, are used in an allele specific extension reaction with biotinylated nucleotides. These products are combined with selected fluorescent bead-oligo combinations. Reaction products are then analyzed on the Bio-Plex instrument, which quantifies the amount of PE signal associated with a particular fluorescent bead address. Each address is linked to a particular oligo which in turn is linked to a particular SNP based on the experimental design. Thus, differences in signal intensity determine which alleles are present in a given input PCR fragment
- Custom Qualitative Ratio Metric Assays: Any experimental design that can be successfully bound to a Fluorescent bead can be used as a direct qualitative ratio metric to identify unique analytes of interest in many varieties of suspensions.
Consultation and Instruction:
Consultation is offered by the Immuno/BioSpot Director, Dr. Christel Uittenbogaart, and Core Manager, Brent Gordon, who can provide protocol workscope planning assistance helpful to investigators who may not have expertise in these assays but wish to incorporate them as a component of their projects. Workshops are offered to investigators for set-up and development of assays and use of acquisition and analysis equipment and software.
Quality Control for the Immuno/BioSpot core is a particular strength. Possessing the very latest and most sophisticated software, the analysis programs utilize advanced algorithms to assess the entire plate and identify trends in spot intensity, morphology, and size among assigned assay groups, accounting for the variation inherent in distinct experimental groups, yet reducing post-analytic, subjective, user alteration throughout Quality Control. Following analysis, variations inconsistent with plate trends are tagged in Quality Control, requesting verification or signaling the necessity to alter counting parameters to retain valuable or unexpected data. Each alteration from the original counting parameters is recorded in the analysis data file and annotated. All altered counting parameters that fall out of the statistical bounds established in the initial assay analysis are also flagged. The software is compliant with 21 CFR part 11 and GLP regulations. Long term project and analysis trends can also be quantified and observed utilizing the SpotMap software. Finally, the Core Manager has extensive, ongoing experience in assay evaluation and image analysis to help the investigator reduce inaccurate data reporting.
The Core's various multiplex software suites combine multiple instrument runs to efficiently analyze multiplex data trends in an investigators biological system. The Core offers the latest and most acclaimed multiplex database applications to effortlessly organize large volumes of data and combine xMAP result files into manageable projects and reports.
Labeled samples can be adjusted and amended with all biologically relevant information for quick data set comparisons, creating error-free custom tables of any size for further processing in standard spreadsheets and statistical software. Calculations can be automated to promote rapid project results in a finished state with little input and negligible need to perform extensive data auditing.
These data management suites permit effortless and accurate automated calculations to test new hypotheses and adjust for new insights and observed trends with GLP compliance. The Core's multiplex software suites offer tools to import and modify proprietary files into universal statistical data and options to rapidly present results in a manner relevant to research and instantly update graphs with modified comparisons when alternate or novel analysis options and data are applied. Graphs and unique spreadsheets can be designed in real time without repetitive copy and paste to Excel. Additionally the multiplex software suites are compliant with 21 CFR part 11 and GLP regulations.
Once the workscope is determined, the investigator contacts the Core manager, Brent Gordon, who is responsible for all Immuno, BioSpot, and Bio-Plex acquisitions, analyses, materials, and equipment. Brent Gordon can help with study implementation, and schedule time and location to meet the investigator for assay consultation, data acquisition, assay analysis, or material provisions. With two distinct campus locations (Factor 12-541 and 188F BSRB), each featuring different analyzers, accessibility is not a problem. Mr. Gordon may be contacted at both locations from the same phone number and e-mail, and requests 24hr advanced notice to schedule or alter an appointment time to help ensure testing is performed in a timely manner. Dr. Uittenbogaart will assist, as requested, in data interpretation during the project and at the end of the studies.