Enzyme-linked immunosorbent assays (ELISAs) allow users to rapidly and sensitively determine the presence and concentration of a target molecule in a biological sample. In this webinar, we discuss the different ELISA formats and ELISA reagents, as well as core ELISA principles and troubleshooting techniques.

Learning Objectives:

• General ELISA principles
• The differences between types of ELISAs, including direct ELISA, competitive ELISA, sandwich ELISA, and SimpleStep ELISA®
• What could go wrong and how to troubleshoot.

Listen in as our scientific expert, Tom Ruyle, discusses how to optimize your IHC  protocol and reviews top troubleshooting tips.   

• How to choose an antibody for IHC
• How to optimize your protocol for the best data
• How to troubleshot poor results

The detection of circulating molecular biomarkers is useful for monitoring pathogenic processes and response to therapeutic intervention1,2. In cancer, genomic and proteomic biomarkers are used to assess tumour sub-type and stage, and determine patient stratification. Research has shown that using a combined signature of multiple biomarkers can better account for patient and epidemiological heterogeneity, and provide a more accurate indication of patient health2. In addition, the desire to leverage minimally-invasive samples has necessitated the development of technologies that can profile biomarkers directly from biofluids such as plasma, serum, and urine. To address this need, we developed the FirePlex Technology platform, which allows for the sensitive and accurate multiplexed detection of protein analytes or miRNAs directly from a wide array of biological samples. The platform uses patented, multi-functional hydrogel particles that allow for in-well multiplexing and panel design flexibility.

For the detection of protein analytes, FirePlex immunoassays use high-performance matched antibody pairs that provide minimal cross-reactivity between individual analytes, up to 5 logs dynamic range, and single-digit pg/ml sensitivity, while requiring only 12.5 µl biofluid input. Similarly, the FirePlex miRNA assay can reliably detect miRNAs directly from as little as 10 µl of biofluid without the need for RNA purification. This assay utilizes single¬ step RT-PCR signal amplification with universal primers, thus leveraging PCR sensitivity while eliminating an independent reverse transcription step and mitigating amplification biases introduced by target¬-specific qPCR.  

FirePlex assays are validated across a wide range of biological samples including plasma, serum, urine and cell culture supernatant. The 96-well plate assay format enables high-throughput sample screening with readout on standard flow cytometers, thereby omitting the need for complex and expensive dedicated instrumentation. Finally, the integrated FirePlex Analysis Workbench software enables rapid data analysis, visualization, and export, and includes key features such as standard curves as well as publication-quality heatmaps and graphs. 

Here we present data from studies using the FirePlex Platform to profile miRNAs in cancer. Together, the combination of multiplexed, high¬-sensitivity assays with powerful bioinformatics tools enables rapid discovery and validation of biomarker signatures from liquid and solid specimens

The natural immune responses that patients develop to their own tumors, as well as therapeutic regimens employing the latest anti-cancer monoclonal antibodies, would reasonably be expected to suppress tumor growth. Yet, a perplexing resistance to both is widespread. The ineffectiveness of the immune system to prevail in these situations suggests that cancers possess tactics to evade antibodies that could otherwise eradicate them. 

With a clinical context in mind, we observed that tumor-associated protein-degrading enzymes can diminish, and in some cases negate, cell killing functions by inducing a single clip in a small part of the antibody structure. The structural modification is so subtle that it had not been previously recognized and would not have been anticipated to so profoundly impair the antibody. To be able to visualize and establish if such cleavage occurred in cancer, we had to develop innovative antibodies for that exact purpose. 

The new antibodies readily enabled the visualization of antibody damage when incubated with cancer cells in the laboratory or within tumor tissues obtained from animals or human patients - more importantly - the same antibody tools possessed the additional and remarkable property of restoring the lost functions to the damaged antibodies. The “rescue” of lost function suggested that this could be exploited as a therapy in cases where tumors cause antibody damage in order to evade our immune system. This novel therapeutic strategy represents a potentially new direction in cancer immunotherapy.

This webinar will review:

• A minor proteolytic cleavage at IgG hinge greatly impairs the effector functions of anticancer antibodies
• Proteolytic hinge cleavage of IgGs as a mechanism of tumor evasion of antibody-based immunity
• The potential of “rescue” of lost IgG Fc function as a novel therapeutic strategy for cancer

Learning Objectives:

• Review mechanisms of tumor cells used to evade interaction from anti-cancer antibodies 
• Learn how antibody technology can be used to develop antibodies capable of visualizing subtle, specific modifications
• Learn about a potential novel cancer therapeutic method of ‘’restoring’’ antibodies damaged by tumor-associated protein-degrading enzymes

Thursday, February 2 | Disruption of axonal transport is an early event in several neurodegenerative diseases. For this reason, it is a potential target for drug discovery. Register today to hear Dr Glennon discuss this exciting topic in Alzheimer's research including, how the disruption of axonal transport contributes to the development of Alzheimer's disease, and the mechanisms of axonal transport of the Alzheimer's amyloid precursor protein, and how these are altered in the disease.

Wednesday, February 1 |  This webinar will review recent data on adhesive mechanisms involved in the maintenance of an epithelial state versus the transition to a mesenchymal-like phenotype, as well as discuss a number of studies in support of the concept that EMT contributes to tumor progression. Register today to join Professor Jean Paul Thiery,  Research Director Emeritus at the Institut Gustave Roussy  Comprehensive Cancer Center at Villejuif for this live webinar and Q&A session.

Thursday, January 26 | Emerging evidence suggests that non-coding RNAs can interact with epigenetic modifiers to both recruit and suppress epigenetic mechanisms, and that this functions can be regulated by activity states of neurons. Register today to join Dr Day as he highlights his recent work exploring how non-coding RNA species regulate DNA methylation in neuronal systems, including interactions between non-coding RNAs and DNA methyltransferases and genome-wide characterization of non-coding RNAs that arise from protein-coding genes , and  reviews the functional modulation of non-coding RNAs in memory formation and behavior. 

Review this powerful technique, get advice on selecting antibodies, and learn troubleshooting techniques with our Epigenetic specialist.

Review Tips and Techniques on how to develop your own ELISA, including:

• Why you would use ELISA assays
• Different types of ELISA assays
• Key reagents
• Quantitation using standard curve
• Optimization and Troubleshooting

Flow cytometry is a powerful technique that allows rapid and quantitative measurements of multiple parameter on individual cells simultaneously. Sheath fluid is used to transport these cells in single file past optical and electronic sensors to detect physical and biological characteristics.

This webinar will review how multiparameter flow cytometry permits precise characterization of multiple cell subpopulations in complex mixtures, including:

• sample processing
• antibody/fluorochrome selection
• controls

ICC is a method for demonstrating the presence and localization of proteins within individual cells in cell culture. Watch this on-demand webinar presented by our resident immunocytochemistry (ICC) expert to learn about,

• Cell culture and sample preparation
• ICC general staining procedure (fixation and permeabilization, antibody staining)
• Recommended controls
• Fluorescence
• Troubleshooting tips
• Antibody selection​

Discuss common IHC problems and review tips and techniques for your experiments with Dr Carolyn Miazga including,

• sample processing
• antigen retrieval
• signal amplification
• controls
• Troubleshooting tips
• antibody selection

In-Cell ELISA, is a well-accepted assay approach to perform quantitative immunocytochemistry of cultured cells using either a colormetric or fluorometric readout.  Join our In-Cell ELISA specialist as he discusses an introduction to this exciting application, followed by some useful optimization and troubleshooting tips.

Review advice on sample preparation and troubleshooting tips in this webinar presented by our resident Western Blot expert.

Learn how the SimpleStep ELISA® kit provides specific and sensitive quantification of target protein with one simple antibody and sample addition step., including how the kit works, advantages of the SimpleStep ELISA kit over the traditional sandwich ELISA, and data generation and analysis with the SimpleStep ELISA kit.

Listen in as our scientific expert, Miriam Ferrer, Ph.D, reviews tips and techniques to help you easily distinguish what type of cell death is present in your experiment, including morphological events that occur during cell death, biochemical events that occur during cell death, and how to assess these events within your experiment.

Quantification of cellular growth before and after an experiment is an essential technique for cell-based studies. Listen in as our scientific expert, Miriam Ferrer, Ph.D., reviews methods to investigate cell viability and cytotoxicity, and discusses how to optimize your cell-based experiments, including how to choose the most appropriate assay for your experiment.

Join Dr Javier Conde Vancells as he reviews the use of primary and secondary antibody conjugates across multiple lab applications, including a review of direct vs. indirect methods in cellular imaging and flow cytometry and the relevance to multi-color analysis.

Review this popular and powerful method for accurate detection of low abundance proteins or changes in protein expression, presented by Dr Martin Broadstock. Review the application of fluorescent western blotting in research, uses of fluorescent detection, and learn how to avoid the challenges and common pitfalls in the use of fluorescence.

Review the similarities and differences between apoptosis and necroptosis, with an introduction to new tools developed to activate effectors of necroptosis.

Review tips and techniques on how to get the best out of your experiments and overcome the most common technical challenges with our resident IHC/ICC specialist.

Topics reviewed include:

• Sequential staining using antibodies
• Simultaneous staining using antibodies
• Multiple stainings using antibodies and cytomarkers
• Analysis of images and validation

Cancer biologists, pharmacologists and immunologists have historically communicated little with each other due to different traditions in training and a lack of integrated perspectives on cancer. Immunologists have been oriented mainly to biological therapies and they have tended to have less knowledge of genetics and pharmacology.

Conversely, cancer geneticists and pharmacologists have been oriented mainly toward small molecule therapies and they have tended to have limited understanding of immunology or immune-based therapies (other than perhaps passive therapies, such as antibodies).

Listen into this on-demand webinar as Dr George Prendergast as discusses the future of Immunochemotherapy, focusing on the convergence of cross-disciplinary themes.

Review the interplay between cell death and cell metabolism, as well as the mechanisms of cell death during cancer and ischemia with Dr Cristina Muñoz-Pinedo. Topics include:

• Crosstalk between cell death and cell metabolism
• Targeting metabolism to kill cancer cells
• Cell death induced by metabolic stress
• Ischemic cell death

Lung stem and progenitor cells are instructed to produce lineage-specific progeny that are regulated by unknown factors in their microenvironment.  Researchers use murine genetic models to identify the functional roles of critical stromal cell populations that control endogenous lung stem and progenitor cell populations, as well as to identify the key signaling pathways that operate between epithelial and stromal cells in vivo.

Recently, ex-vivo organoid co-culture systems have been developed, which allow researchers to govern the lineage specification of epithelial stem/progenitor cells, and discover a new regulatory signaling pathway (BMP4-calcineurin/NFATc1-TSP1) that operates between damaged epithelial cells and endothelial cells to derive the alveolar lineage specification of distal lung stem cells-  bronchioalveolar stem cells (BASCs).

This webinar reviews:

• Lung organoid co-culture system as the modelling of cell-cell interactions
• Crosstalk between stromal/niche cells and lung stem/progenitor cells for lineage specification
• The future of lung stem cell-mediated epithelial homeostasis and related pulmonary disease research

Join Dr Ann Wheeler of Queen Mary University as she reviews the basic principles of confocal microscopy, including:

• Sample preparation for confocal microscopy</li><li>When you need a confocal microscope
• Advantages over epifluorescent microscopy</li><li>How the confocal microscope works
• Pitfalls with confocal microscop
• Advanced applications

This webinar is ideal for anyone who is interested in carrying out confocal microscopy or is currently using confocal microscopy, as well as anyone who would like to better understand the technique and how it can be applied to their experiments.

Bivalent domains are thought to be controversial, yet crucial,  regulatory hubs in the differentiation of stem cells. Listen in as  Dr Philipp Voigt of the Wellcome Trust Centre for Cell Biology at the University of Edinburgh as he reviews the concepts of bivalent domains, the impact of nucleosomal asymmetry and bivalency, and current views on the functions and mechanisms of bivalent domains.

Listen in as our scientific expert, Miriam Ferrer, Ph.D., reviews why apoptosis is a key process in ensuring normal development and tissue functionality in multicellular organisms, and discusses which assays to use for qualitative and quantitative analysis of apoptotic markers.

• Identification of scFv antibody fragments using phage display
• Expression, affinity maturation, and characterization of scFvs 

Register today to hear more about this exciting topic, and have your questions answered during a live Q&A session!

Learning Objectives:

• Learn about a potential strategy for targeting neo-antigens for cancer therapies
• Learn one use of phage display technology for developing therapeutic agents against cancer

Antibodies are tremendously useful and versatile tools and they have found use in many areas of life science research. However, antibodies can be difficult to make and are used in inherently complex biological systems. Consequently, they can be prone to quality issues: batch-to-batch variability, lack of specificity, or poor sensitivity. The impact of this can be substantial, both financially and scientifically as identified in recent studies.
 
Learn how at Abcam, we are raising industry quality standards by using newer and improved technology to manufacture and validate our products. Addressing both specificity and reproducibility issues, we have recently introduced knockout (KO) validation at scale. Utilizing human *KO cell lines generated from haploid cellular models using CRISPR/Cas9, the technology has enabled the development of a growing range of KO validated antibodies, including many recombinant monoclonals.
 
This webinar will review:  

• How antibody manufacture affects quality
• Antibody validation methods 
• Abcam’s knockout validation initiative
• Best practices to ensure reproducible results

Learning objectives:

• Understand why antibody quality is affecting life science research
• Be aware of the criteria to assess when selecting an antibody for your research
• Understand how Abcam is using CRISPR/Cas9 modified cells to validate the specificity our antibodies

*Abcam validates antibodies using KO cell lines from Horizon Discovery