Antibody Insider—Issue 2017 Q2
Immunotherapies, which refocus an individual’s immune system to better fight disease, have shown promise in the treatment of cancer as well as other diseases. Current immunotherapy approaches include cytokines (protein or antibodies directed to specific cytokines), cancer vaccines, checkpoint proteins, and immune cell adaptive transfer.
Immune checkpoint therapy uses monoclonal antibodies that recognize proteins called immune checkpoints. Checkpoint proteins are either inhibitory or stimulatory, as they are involved in stopping cell signaling, which results in T cell exhaustion, or promoting the activation of T cells, respectively. In these therapies, monoclonal antibodies against the inhibitory proteins are designed to “release the brake” so that cells of the immune system (henceforth referred to as “immune cells”) see the cancerous cells as “bad”, allowing for their elimination. The first approved checkpoint therapies for melanoma are based on antibodies that recognize CD152 (CTLA-4), CD279 (PD-1), and CD274 (PD-L1), and their efficacy is being tested with other cancers. Current treatments are now looking to combine therapies such as PD-1 and PD-L1. Although T cells play a key role (especially CD8+ T cells), more data concerning the recruitment role by macrophages and DCs are also coming to light. In fact, in addition to the CD8 T cells’ killing properties, the roles of myeloid derived suppressor cells (MDSCs), NK cells, and M1 (tumor rejecting phenotype) macrophages are shown to be contributors in responsiveness to therapies.
Understanding the phenotypes of immune cells and hence proteins involved in fighting cancer is critical for studying these potential therapies, and several techniques are emerging as valuable in this research. Flow cytometry is an extremely powerful cell analysis technique for both cells in circulation and samples derived from solid tumors, able to deliver information on phenotypes as well as cell health (proliferation vs. apoptosis) using a mutiparametric approach. Multicolor immunohistochemistry (IHC) allows the discrimination of cells’ phenotypes and locations within a solid tumor. IHC studies have shown that when CD8+ T cells are found in close proximity to PD-L1+ macrophages, tumors are more responsive to checkpoint inhibitor therapies. Lastly, assays such as ELISA/ELISPOT or multiplex immunoassays (such as ProcartaPlex assays) allow the monitoring of secreted and shed proteins in patients. Cytokines and other proteins like IFNg, perforin, and granzyme B are examples of routinely monitored proteins.
Whether your research is focused on searching for and detecting the new immune checkpoints or exploring the novel functions of immune checkpoints, Thermo Fisher Scientific offers a wide range of research antibodies to help ensure your success, including over 1,000 antibodies targeting immune checkpoints for both human and mouse experiments. Our experienced custom service team can also save you time by developing antibodies to meet your specifications. For more detailed antibody product descriptions, see Immune Checkpoint Antibodies
ProcartaPlex: Human Immuno-Oncology Checkpoint Panel
The ProcartaPlex Immuno-Oncology Checkpoint Panel is the first commercially available multiplex immunoassay system that allows the simultaneous detection of the soluble forms of the following analytes: BTLA, GITR, HVEM, IDO, LAG-3, PD-1, PD-L1, PD-L2, Tim-3, CD28, CD80, 4-1BB, CD27, and CTLA-4. Soluble isoforms, or shed variants of these molecules, have been described and can be quantitated by this innovative assay.
The assay’s capture antibodies are covalently bound to the surface of 6.5 µm microspheres that are internally dyed with precise proportions of red and infrared fluorophores. The distinct blends of these two fluorophores (called “bead sets”) result in unique spectral addresses that can be detected when analyzed on Luminex platforms. As with a traditional immunoassay, antigen quantification is enabled by a fluorescently labeled secondary antibody whose signal intensity is proportional to the concentration of protein detected. For multiplexing purposes, a specific bead set is assigned to each analyte, enabling the simultaneous measurement of multiple analytes from a small sample volume (25 µL for serum or plasma samples, 50 µL for cell culture supernatant). The technology allows for the simultaneous detection of multiple soluble immune checkpoint markers using highly specific antibody pairs to create bead-based sandwich immunoassays.
Advantage of the approach
The multiplex immunoassay for the detection of soluble checkpoint markers provides information complementary to the data obtained by pathologists from tumor tissues. The analysis of protein from liquid biopsies (i.e., plasma or serum) is easy and convenient, minimally invasive, and not biased by the location of the sampling like tissue biopsies are. In addition, the ability to monitor soluble checkpoint molecules, correlate these levels with the progression of disease in longitudinal studies, and begin to associate biomarker levels with checkpoint blockade therapy response, is a highly promising area of application of the Human Immuno-Oncology Checkpoint Panel.
Quantification of soluble checkpoint molecules is a promising new tool that can also be combined with the assessment of cytokines and chemokines in multiplex immunoassays to support research in the field of immuno-oncology and cancer immunology.
Learn more about the ProcartaPlex Human Immuno-Oncology Checkpoint Panel
Standard curves for the human ProcartaPlex Immuno-Oncology Checkpoint 14-Plex Panel.
Thermo Fisher Scientific maintains a portfolio of over 74,000 Invitrogen antibodies, recognizing over 85% of human targets. One key focus has been to provide a set of antibodies suitable for immunohistochemistry (IHC). With the recent integration of the eBioscience antibody portfolio, more than 10,000 of our antibody products display IHC data along with other product data on our website, and we are adding new images regularly as we test new sample types. The ability to visualize protein expression within tissues is an important tool in understanding the biology associated with targets. By using markers for specific cell types, IHC can be used to identify specific cell types within tissue samples. By using specific markers for disease states, IHC can be used to study cancer, infections, and other diseases.
ALDH1L1 Monoclonal Antibody (7G8), Biotin, eBioscience (Cat. No. 13-9595-82). Immunohistochemistry of formalin-fixed, paraffin-embedded human cerebellum using 20 µg/mL Mouse IgG2b K Isotype Control Biotin (left) or 20 µg/mL Anti–Human 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) Biotin (right), followed by streptavidin-HRP and DAB visualization. Nuclei are counterstained with hematoxylin.
Antibody choices for fluorescent and chromogenic IHC
IHC using a chromogenic substrate like DAB has been routinely used for examining tissues. Chromogenic IHC is especially suited for archival storage of samples, allowing scientists to look at the tissues months or even years later. Fluorescent staining allows for multiplexing, which facilitates colocalization studies. Because the fluorescent signals dissipate over time, fluorescent IHC is normally not used for archival storage. Whether used with HRP secondary antibodies and a colorimetric substrate or with fluorescent labels like the Alexa Fluor dyes, Invitrogen antibodies are developed to provide specific staining with expected localization and expression levels.
Anti–α-tubulin antibody (Cat. No. A11126). A 14 µm coronal section of mouse hippocampus stained with an anti–α-tubulin antibody (Cat. No. A11126) labeled using the Zenon Alexa Fluor 488 Mouse IgG1 Labeling Kit (Cat. No. Z25002). The section was counterstained with the NeuroTrace 530/615 red fluorescent Nissl stain (Cat. No. N21482) to visualize neuronal cell bodies, and Hoechst 33258 (Cat. No. H1398, H3569, H21491) to stain nuclei.
Building reliability into our antibody products
Success with IHC requires use of the correct samples, correct preparation of those samples, and a high-quality specific antibody. During development of an antibody product, our scientists identify and test tissues that exhibit normal or abnormal expression of the target, including brain, heart, muscle, intestine, and others in healthy and various disease states. When specific antigen retrieval is required, that information is reported in the product manual and on the web pages. Finally, when possible, the antibodies are tested in multiple applications to show that the antibody not only works in IHC but is verified to bind the correct target by other methods as well. Recently we have begun to analyze the direct interaction of antibodies and target proteins by mass spectrometry, one of the few methods that provide amino acid sequence evidence of antibody specificity.
Find your antibody on our website at thermofisher.com/antibodies. Results can be filtered by application for those antibodies that have been shown to work on paraffin-embedded or frozen samples.
New Invitrogen eBioscience Super Bright 645 and 702 antibody conjugates
Discover the bright spots with the newly expanded eBioscience Super Bright antibody portfolio. As with other Super Bright antibody conjugates, Super Bright 645 and Super Bright 702 conjugates provide exceptionally bright readouts and are compatible with flow cytometers with a violet laser (405 nm). We have also released many new clones conjugated to Super Bright 436 and Super Bright 600, increasing available targets and formats.
New Super Bright 645 is a tandem dye consisting of Super Bright 436 and an acceptor dye with an emission peak of 645 nm. It can be detected using a 660/20 bandpass filter or equivalent, similar to eVolve 655. This tandem polymer dye offers brightness similar to or better than that of Brilliant Violet 650. Super Bright 645–conjugated antibodies typically demonstrate less spillover into other violet channels.
Our fourth dye, Super Bright 702, comprises Super Bright 436 and an acceptor dye that has an emission peak of 702 nm. It can be detected using a 710/50 bandpass filter or equivalent, similar to Brilliant Violet 711. Super Bright 702–conjugated antibodies are similar in brightness to Brilliant Violet 711 and exhibit reduced compensation out of blue laser channels and less spillover into the Brilliant Violet 786 channel.
Super Bright polymer dyes are optimized for use in flow cytometry and may allow for better discrimination of dim populations due to their brightness. Fully compatible with other commonly used fluorescent molecules, formaldehyde- and methanol-based fixatives, and UltraComp eBeads microspheres, the Super Bright conjugates allow for ideal flow cytometry multicolor antibody panel design.
Super Bright dyes can be used in flow cytometry applications in the same way that you use traditional fluorophores. However, if two or more Super Bright dye–conjugated antibodies are combined in the same panel, the use of Super Bright Staining Buffer (Cat. No. SB-4400-42) is recommended to minimize any nonspecific interaction that may occur between these polymer-based dyes. Super Bright Staining Buffer is formulated for 5 µL/test, making it convenient for use in master mixes and cocktails.
As we continue to expand our portfolio with new clones and additional formats, Super Bright dyes provide more choices for multicolor panel design and allow you to expand the utility of your violet laser. eBioscience Super Bright antibody conjugates are the user-friendly alternative you have been waiting for.
Super Bright 645 antibody conjugates offer brightness similar to or better than that of Brilliant Violet 650–conjugated antibodies, with comparable or reduced background. (A) Mouse splenocytes were stained with CD8a (53-6.7) conjugated to either Super Bright 645 (red histogram) or Brilliant Violet 650 (grey histogram), using the same concentration of antibody. (B) Human peripheral blood cells were stained with CD8a (RPA-T8) conjugated to either Super Bright 645 (red histogram) or Brilliant Violet 650 (grey histogram), using the same concentration of antibody.
Super Bright dye–conjugated antibodies improve discrimination of dim antigens. Human peripheral blood cells stained with Anti–Human CD11c (clone 3.9) conjugated to Super Bright 705 (left) or APC (right), and Anti–Human CD20 (clone 2H7) conjugated to PE, at their respective optimal concentrations. (Note: Super Bright 705 is supplied at 0.5 µg/test, and APC is supplied at 0.5 µg/test. 2H7-PE is supplied at 0.06 µg/test.)
Ready, Set, Flow!
Invitrogen Ready Flow reagents are ready-to-use solutions designed to stain your cells for analysis by flow cytometry without calculations, dilutions, or pipetting. These reagents simplify the most common flow cytometry assays, including dead-cell identification and cell cycle analysis. Preparing your samples for flow cytometry analysis is easy.
Cell cycle analysis with FxCycle Violet Ready Flow Reagent and the Invitrogen Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit.
Explore new possibilities in immunoassays
Live webinar, June 6, 2017
Introducing the new Invitrogen ProQuantum High Sensitivity immunoassay kits—the new generation of protein quantitation
Our latest platform innovation provides researchers with a product line of ready-to-use immunoassays to easily measure low-abundance proteins in very small sample volumes using real-time PCR technology.
By utilizing powerful technologies such as TaqMan Assay–based proximity ligation assay (PLA) and SiteClick antibody labeling, we have combined the analyte specificity of our own high-affinity antibody–antigen binding with the signal detection and amplification of real-time PCR, to create an exceptional high-performance assay.
- High sensitivity—detect low levels of proteins with greater sensitivity than with other traditional methods like ELISA
- Small sample consumption—use 2–5 µL of sample (e.g., 2 µL vs. 150 µL for triplicate wells with other methods)
- Fast, easy workflow—no wash steps, 2 hours from sample to answer
- Broad dynamic range—≥5 log units minimizes sample dilutions needed to ensure falling within the range
- No proprietary instrument to purchase—runs on any real-time PCR instrument
- Includes intuitive, free cloud-enabled software—for robust data analysis and statistical groupwise comparison
Standard curve for human IL-8 assay, compared to that of an ELISA. Note the longer sigmoidal standard curve (larger dynamic range) and low-end separation for quantitation of smaller amounts of protein.
Protocol workflow. (A) Two incubation steps and no wash in between.
Learn more and register for the webinar at thermofisher.com/explore.
Introducing the largest screening panel on the market: ProcartaPlex Human Immune Monitoring 65-Plex Panel
The ProcartaPlex Human Immune Monitoring Panel (Cat. No. EPX650-10065-901) enables researchers to analyze 65 soluble protein targets simultaneously in a single well using only 25 µL of sample. This new and powerful assay uses Luminex multiplexing technology, allows researchers to get more data out of limited, precious samples, and expedites the path to research discovery. This higher-plex assay can be analyzed using the Luminex 200 or FLEXMAP 3D instrument. (At this time, MAGPIX instrument users are limited to 50 targets per assay.)
ProcartaPlex assays are subjected to the same stringent and robust development and manufacturing as our extensively validated coated and uncoated ELISA kits, allowing researcher to move from platform to platform and still get consistent results.
65-Plex target list
APRIL, BAFF, BLC, CD30, CD40L, ENA-78, eotaxin, eotaxin-2, eotaxin-3, FGF-2, fractalkine, G-CSF, GM-CSF, Gro a, HGF, IFN-α, IFN-γ, IL-10, IL-12 p70, IL-13, IL-15, IL-16, IL-17A, IL-18, IL-1α, IL-1β, IL-2, IL-20, IL-21, IL-22, IL-23, IL-27, IL-2R, IL-3, IL-31, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, I-TAC, LIF, MCP-1, MCP-2, MCP-3, M-CSF, MDC, MIF, MIG, MIP1α, MIP-1b, MIP-3α, MMP-1, NGF-β, SCF, SDF-1α, TNF-β, TNF-α, TNF-R2, TRAIL, TSLP, TWEAK, VEGF-A
Explore our broad portfolio of preconfigured panels and combinable single analytes and create your own custom panel using our new Luminex Panel Configurator and Selection Tool.
Flow with the experts
Do you work with a flow cytometer and want to learn more?
Check out the flow cytometry articles on the Behind the Bench blog. Learn from experts about flow cytometer fluidics or getting the most from your cell proliferation assay.
(Left to right) Dr. Greg Kaduchak, Dr. Andrew Filby, and Dr. Bill Telford. All are contributors to the Behind the Bench blog for flow cytometry.
Optimized multicolor immunofluorescence panels (OMIPs)
Jump-start your flow cytometry panel design with published antibody and reagent panels. Read about Optimized Multicolor Immunofluorescence panels (OMIPs)—published panels optimized for flow cytometry—in the recently released BioProbes 74. Each OMIP publication contains an overview of the purpose of the panel, a listing of antibody clones and fluorophore combinations, information on sample type and similarities to any existing OMIPs, and a figure showing representative experimental results, including the gating scheme. Download the article and check out the resource page today.
Example of immunophenotyping using OMIP-009.
Meet our antibodies
Introducing the new Meet Your Match video, now live on thermofisher.com. Join Antigen, Secondary HRP, and Primary as game show contestants looking for their ideal matches to create great westerns.
Our antibodies are seeking partners for binding relationships. Meet all of our antibodies and watch their videos live on: thermofisher.com/meetourantibodies
Read and sign up to receive BioProbes journal
BioProbes Journal is a biannual publication that highlights a wide range of Thermo Scientific and Invitrogen cell biology products and applications. From new reagents and technologies to product reviews and online tools, we keep you up to date on the latest breakthroughs in cell and protein analysis.
See the latest issue at thermofisher.com/bioprobes
New eLearning courses for protein and cell analysis applications
We are pleased to announce the launch of a series of eLearning courses aimed at basic research topics within protein and cell analysis. Providing succinct, contextual information, these narrated courses include:
- Animated diagrams
- Knowledge-check questions
- Certificate of completion and badge
- Downloadable course materials
- Relevant supplementary resources
To learn more, visit thermofisher.com/elearningcourses
Introducing the new PCA Education site
The Protein and Cell Analysis (PCA) Education site is a free-access virtual learning environment dedicated to showcasing rich educational content that represents our Protein & Cell Analysis and Antibodies & Immunology portfolios.
To learn more, visit thermofisher.com/proteincelledu
Join us for our PCA Education kick-off event—May 30, 2017
Antibody Validation Forum
Addressing the antibody reproducibility crisis: A panel discussion with key scientific leaders
Join a live, virtual panel that includes members from the International Working Group for Antibody Validation (IWGAV). These industry thought leaders will describe new standards devised for optimizing antibody validation methods that emphasize antibody specificity verification. Please come with questions for our panelists and learn more about this exciting initiative.
- Anita Bandrowski, PhD, CEO of SciCrunch, Inc.
- Aled Edwards, PhD, CEO of the Structural Genomics Consortium
- Paul Wallace, PhD, Director the Flow and Image Cytometry Facility, Roswell Park Cancer Institute
- Matt Baker, John Rogers, PhD, Christoph Hergersberg, PhD (moderator), Thermo Fisher Scientific
Following the panel forum is a webinar series presented by scientists from Thermo Fisher Scientific
- John Rogers, PhD, presents: Antibody validation with immunoprecipitation/mass spectrometry and orthogonal methods
- Castle Funatake, PhD, presents: Super Bright antibody conjugates for flow cytometry—bright polymer dyes for the violet laser
- Emily Halbrader presents: Chemiluminescent western blot detection: bright and bold detection no matter how scarce your target
- David Bourdon, PhD, presents: The new generation in immunoassays
To learn more and to register for the panel forum and the webinar series, visit Antibody Validation Forum.
CiteAb names Invitrogen secondary antibodies as the 2017 winner of the Secondary Antibody Company of the Year Award
This award recognizes Thermo Fisher Scientific and its Invitrogen secondary antibodies “as the most successful antibody company at providing secondary antibodies based on CiteAb data”. CiteAb, in awarding this distinction, looked for the company with the most citations for secondary antibodies, from 2016 data. CiteAb Awards celebrate the very best suppliers and individuals in the research antibody sector worldwide.
This is the first year for this new category, and we couldn’t be more proud of this industry recognition.
Antibodies are some of the most critical reagents in life science and medical research. Over the past several years a number of high-visibility publications and forums have drawn more attention to the need to develop higher standards for validating and ensuring antibody reproducibility. As we spoke to researchers, it became clear that the industry needed to act to serve their needs and concerns. In September 2015, Thermo Fisher Scientific provided financial support to help organize a group of leading researchers from institutions throughout the world to draft a set of proposed standards for the best ways to validate antibody specificity.
The International Working Group for Antibody Validation (IWGAV) published their proposed strategies in Nature Methods in September 2016. In the fall of 2016, many of the IWGAV members as well as representatives from antibody producers, pharmaceutical companies, and leading academic research labs met at an event coordinated by the Global Biological Standards Institute (GBSI) to further study this topic. This meeting reviewed and validated the IWGAV’s proposed standards and discussed ways to implement the standards across the industry.
As a result of our active discussions and key participation in these forums, Thermo Fisher Scientific has seen a clear consensus develop around several impactful practices to help further advance antibody validation.
We are excited to present the new Thermo Fisher Scientific validation guidelines that we will follow in our ongoing efforts to further validate the Invitrogen antibody portfolio. These new guidelines will add to the extensive application validation already performed for Invitrogen antibodies, and will focus on confirming antibody specificity. Testing antibodies for functional validation is critical and must be carried out with the utmost rigor, but it can be a further challenge to confirm antibody specificity. By combining both functional validation and specificity testing, we aim to ensure that antibodies meet the highest possible standards of overall antibody validation.
Thermo Fisher Scientific is working to test our portfolio of antibodies using the new validation testing methods. Mass spectrometry is used to identify the physical amino acid sequence of the protein of interest as well as any other nonspecific binders and known protein–protein interactions. This powerful technique is completely unique in its ability to provide the sequence of the protein the antibody detects and will help us further elucidate antibody-binding interactions. See some examples of this testing.
For genetic modification, Thermo Fisher Scientific has deployed the use of CRISPR/Cas9 as well as RNAi to provide controls that confirm antibody specificity versus the wild type. See the workflow and examples of this powerful technique.
These are just a few examples of key tools that Thermo Fisher Scientific is using to systematically validate the Invitrogen antibody portfolio for specificity. Invitrogen antibodies have more than 100,000 supporting images and more than 60,000 reference citations. Stay tuned as Thermo Fisher Scientific continues to establish new standards in antibody testing and characterization.
*The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.
New Alexa Fluor Plus packaging helps save time and space
In our last edition we announced the new Alexa Fluor Plus secondary antibodies, with higher signal-to-noise ratios and superior sensitivity to help you detect low-abundance targets. But that’s not all. We have developed new packaging that helps you save time and space:
- Storage made easier—the new vials have flagless labels; no more struggling to wrap the flag around the vial to fit into racks or centrifuges
- Identification made easier—vials have colored caps corresponding to the conjugated Alexa Fluor Plus dye; so, for example, if you are looking for an Alexa Fluor Plus 488 antibody, just look for the green cap
- Easier to see how much antibody you have left—vials are clear so you can easily see the volume remaining; no need to pipet from an opaque vial just to estimate how much antibody remains
We showed real scientists the incredible images being created using new Invitrogen Alexa Fluor Plus secondary antibodies.
Check out this video to see their reactions:
Now watch our videos addressing your questions on phosphospecific antibodies
Antibody character stickers everywhere!
Our popular antibody characters are now available in a collectible card format, each featuring a peel-off character sticker. There are three poses for each character, as well as a group pose, so collect them all. Request these collectible cards from your sales representative, and post your photos with them on the Invitrogen Facebook page!
For Research Use Only. Not for use in diagnostic procedures.