Immune Monitoring Services

Comprehensive Characterization of the Immune Response

The Immune Monitoring shared resource provides a full suite of services to monitor and characterize immune responses. We offer support for the development and testing of new CAR T cells, including product-release testing of newly engineered T cells for Phase 1 clinical trials. As part of this effort, our tetramer and molecular assays teams provide services to the Hutch's Therapeutic Products Program. The IM team also works with investigators to develop new assays and bring in cutting-edge new technologies. 

To request services, please refer to our rates and scheduling page.

Certain instruments are open to all investigators on a first-come, first-served basis and no reservation is currently required.
 

ON THIS PAGE

Tetramer Production  |  Cytokine Assays  |  Molecular Assays  |  Monitoring and Phenotyping Immune Cells  |  Molecular Cloning by DNA Assembly  |  Equipment

Schedule With Us

To schedule services, on-site assistance or training with the Immune Monitoring, or to get more information about how we can work with you, contact the IM team lead:

Jianhong Cao, Ph.D.
Director/Senior Staff Scientist

Tetramer Production

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Research-Grade Tetramers

We produce Class-I tetramers and monomers for research use. Recombinant Class-I heavy chain and β2-microglobulin are expressed in E. coli. The MHC monomeric complex is refolded with epitopic peptides in vitro. After biotinylation, monomers are conjugated to streptavidin to form tetramers.

High-Grade MHC Class-I Tetramers

In a separate production line in a clean room, we produce tetramers in a singular procedure and sterilized them at the end. Access more information and a complete list of the MHC Class-I alleles and fluorochrome-conjugate options available for tetramer production by downloading this PDF:

Director Dr. Jianhong Cao advises lab technicians.
Director Dr. Jianhong Cao advises on an FPLC run. Photo by Robert Hood / Fred Hutch News Service
Barcoded Tetramers

We produce tetramers with a DNA barcode, which can be used for various single-cell sequencing applications, including CITE-seq. Please contact our team to request use of the instrument.

Open Conformer Tetramers

Certain Class-I tetramers will be produced without peptide. Alternatively, we can also produce certain tetramers with UV-cleavable peptides.

MHC Class-II Tetramers

We are in the process of establishing MHC Class-II tetramer production.

Fast Protein Liquid Chromatography

We use fast protein liquid chromatography, or FPLC, to purify and analyze proteins. Depending on the type of the column used, proteins are separated based on the properties of size, charge and hydrophobicity. Our FPLC instrument is used mainly for pMHC tetramer production. Upon request, we can also run other protein samples. We have three FPLC instruments available, including one reserved only for clinical-grade tetramer production.

This is not generally a shared instrument; please contact our team to inquire about use.

Scientific piece of equipment
Photo by Robert Hood / Fred Hutch News Service

Cytokine Assays

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Quantitative Assessment of Cytokines and Other Analytes

We perform quantitative immunoassays on your biological samples to give you concentrations of various cytokines and other analytes in serum, plasma and cell culture supernatants.

To learn more about our library of common cytokines, as well as other proteins we develop at researchers’ request, please refer to our FAQ:

Scientific piece of equipment
Photo by Robert Hood / Fred Hutch News Service
Single-Cell Cytokine Analysis

We offer automated single-cell analysis of cytokines via the IsoLight system from IsoPlexis. The platform is designed to understand and characterize differences among single cells. By mapping thousands of cells per sample, the system can reveal full functional profiles and polyfunctionality among cell subsets to determine patient response and product quality. 

Please contact our team to request use of the instrument.

Isolight Instrument
Photo by Robert Hood / Fred Hutch News Service
Ultrasensitive Cytokine Analysis

The SR-X Ultra-Sensitive Biomarker Detection System from Quanterix enables ultra-sensitive biomarker detection. The SR-X allows multiplex detection of up to six analytes per well, with low volume requirements to increase productivity and throughput. An extensive menu of Simoa assay kits are available to measure critical biomarkers with substantially higher sensitivity than standard immunoassay methods, enabling detection of both normal and acute levels with high precision across a range of sample types. 

Please contact our team to request use of the instrument.

Quanterix Instrument
Photo by Robert Hood / Fred Hutch News Service

Molecular Assays

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Clonal TCR and CAR-T Analysis and Tracking

The unique feature of each T-cell clone is found in its recombined T-cell receptor sequences. We can identify the clonal TCR β-chain sequence by real-time PCR and direct sequencing of the clonal cell sample. The clonal TCR-β sequence can be cloned and used for quantitative PCR analysis.

With knowledge of the clonal TCR sequence, specific primers and a probe for quantitative RT-PCR analysis can be designed, allowing the quantitation of the clonal T cells in a cell population. We provide this service in the setting of adoptive T-cell transfers in immunotherapy trials for cancer patients. We also provide custom-designed qPCR assays for CAR T-cell therapy trials.

ImmunoSEQ Assay

The immunoSEQ Assay from Adaptive Biotechnologies uses a multiplex PCR-based assay to sequence directly from genomic DNA. immunoSEQ provides accurate, quantitative and high-throughput sequencing of the CDR3 variable chain of immune-cell receptors. This service is supported by the Genomics & Bioinformatics shared resource.

TapeStation

We offer the Agilent 4200 TapeStation for DNA analysis. This includes high-sensitivity analysis of DNA from 100 to 5000 base pairs. Please contact our team to request use of the instrument.

Scientific piece of equipment
Photo by Robert Hood / Fred Hutch News Service
Qubit 4 Fluorometer

The Qubit 4 Fluorometer from Thermo Fisher Scientific is designed to accurately measure DNA, RNA and protein quantity, as well as RNA integrity and quality, using the highly sensitive Qubit assays. 

Please contact our team to request use of the instrument.

Qubit instrument
Image courtesy of Thermo Fisher Scientific
TCR Spectratyping

Spectratyping is a set of multiplex RT-PCR reactions that amplifies all T-cell receptor beta-chain families to reveal the clonal-length polymorphism in the CDR3 region. This technique is used to access clonal composition in T-cell populations. TCR spectratyping is often used to determine clonal expansion, contraction and deletion during immune response and reconstitution. It can be performed on PBMC samples or FACS-sorted cell subsets.

Mason Lai prepares DNA in a lab.
Immune monitoring specialist Mason Lai prepares DNA for qPCR to measure T-cell persistence in an ongoing clinical trial. Photo by Robert Hood / Fred Hutch News Service

Monitoring and Phenotyping Immune Cells

Characterization of Immune Cells

We aid investigators seeking to monitor and phenotype immune cells according to differentiation, activation, functional markers and other characteristics. Our technologies and capabilities allow users to characterize cell-surface and intracellular proteins, detect antigen-specific T cells, and isolate specific cell subsets for further analysis. We provide flow cytometry panel development, analysis and data presentation in accordance with specific study requirements.

Our capabilities are supported by close connections to other Fred Hutch shared resources, including the Flow Cytometry core. We also work with users to characterize cells stored with the Specimen Processing & Research Cell Bank shared resource.

Scientific piece of equipment
Photo by Robert Hood / Fred Hutch News Service
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Molecular Cloning by DNA Assembly

BioXp 3200 System

The robotic BioXp 3200 system workstation builds double-stranded DNA fragments and plasmid clones from custom-designed reagents. Gibson Assembly is the underlying molecular technology. We order designed DNA sequence reagents from CODEX DNA, which will usually be delivered in three to five business days. Our staff will build the DNA overnight on the BioXp.

The following are available:

  • dsDNA fragments: DNA fragments between 300 and 1800 base pairs can be assembled. Up to 32 fragments can be assembled in each BioXp overnight run.
  • Cloning: DNA fragments can be cloned into your vector of choice. The customer provides the linearized vector (up to four different vectors per run) to our lab and a Gibson Assembly reaction takes place on the BioXp with your assembled fragments. The customer can then transform the potential clone into competent E. coli cells and verify the constructs by sequencing.
  • RapidAMP (under development): Eliminate the transformation step by amplifying your clonal plasmid DNA for direct use in transfection. Up to 24 constructs can be built with up to four different vectors in each run.
Scientific piece of equipment
Photo courtesy of Codex DNA

Equipment

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BioTek Synergy H4 Hybrid Reader

The Synergy H4 with Hybrid Technology is a multi-mode detector for reading 96-well dishes. The reader has quadruple monochromator-based optics and filters that provide flexibility in usage. Users can select any wavelength, from the low UV to the near-infrared. Detection methods include fluorescence, time-resolved fluorescence, fluorescence polarization, AlphaScreen/AlphaLISA, luminescence and UV-visible absorbance. Read methods include end-point, kinetic, spectral scanning, and well-area scanning. The instrument can read plate formats from six to 384 wells.

Scientific piece of equipment
Photo by Robert Hood / Fred Hutch News Service
CTL ImmunoSpot Analyzer

This ImmunoSpot reader from CTL is specially designed for reading ELISPOT and FluoroSpot plates. The image-acquiring and analysis system is equipped with a microscope that can scan 96-well plates mounted on a workstation. The accompanying software enables the recognition and quantitation of immunospots in each well. This flexible system can read different plates from various suppliers either automatically or manually, with many options for manipulation.

Scientific piece of equipment
Photo by Robert Hood / Fred Hutch News Service
Berthold Model Centro LB 960 Microplate Luminometer

The DLR-ready Berthold Model Centro LB 960 microplate luminometer has three injectors.

Scientific piece of equipment
Photo courtesy of Berthold Technologies
Roche LightCycler 480 II

The Roche LightCycler 480 performs quantitative real-time PCR analysis as well as methylation and high-resolution melting analyses.

Scientific piece of equipment
Photo courtesy of Roche
Hidex Sense

Hidex Sense is a scintillation and luminescence counter reads 96-well plates. It is commonly used for the 3H-thymidine incorporation lymphoproliferation assay and chromium-51 release assay. It also reads luminescence for many reporter assay systems.

Scientific piece of equipment
Photo by Robert Hood / Fred Hutch News Service

Questions about our immune monitoring services or how to schedule with us?