Overlooked benefits of using polyclonal antibodies.

The benefits of polyclonal antibodies as tools for assay-specific target discovery and detection are numerous. As the future of basic research, diagnostics and biomarker discovery is dependent on high-quality reproducible data, there is a need to understand the importance and benefits of these valuable tools. All antibody forms - polyclonal, hybridoma-based monoclonal and recombinant monoclonal - have pros and cons for development, validation and use. Yet, polyclonal antibodies are embroiled in a firestorm of controversy concerning data reproducibility. We address best practices for developing and using polyclonal antibodies, pitfalls to their use and how to avoid them, and benefits to the life science community. Eliminating their use risks overlooking the unique benefits of polyclonal antibodies as 'fit-for-purpose' life science tools.

Express γ-H2AX Immunocytochemical Detection of DNA Damage.

DNA can be damaged by many environmental factors including chemical agents and ionizing radiation which induce the formation of DNA double-stranded breaks (DSBs). If DSBs are not repaired in a timely fashion this may cause the disruption of genome integrity, which can result in cancer development. Typically, DSBs are followed by phosphorylation of histone protein H2AX, a member of the H2A family. Immunocytochemical detection of phosphorylated H2AX (e.g., γ-H2AX) appears to be a useful technique for assessing DNA damage. Such an assessment is easy to do by analyzing labeling for γ-H2AX under the microscope and does not require an expensive laboratory setup. Using HeLa cells treated with camptothecin as a model, we developed an easy-to-run protocol to analyze DSBs. Our protocol can be applied to testing the potency of different chemicals to induce DSBs in different types of cells and requires around 2 h to complete.

Structural insight into allosteric modulation of protease-activated receptor 2.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

Phenotyping CD4+ hTh2 Cells by Flow Cytometry: Simultaneous Detection of Transcription Factors, Secreted Cytokines, and Surface Markers.

Flow cytometry is a powerful technique that allows simultaneous detection of multiple markers on a specific cell population. This method is virtually unlimited as long as the specimen of interest can be put into a single-cell suspension for staining and subsequent analysis by the flow cytometer. Most investigators using this methodology are doing so because their cell population is rare in frequency and requires multiple markers to characterize their population of interest; thus standard methods such as Western blot and IHC are unsuitable due to limitations in cell number and the number of markers available. Most investigators using this method are using 6-14 parameters to study their cell populations of interest: however, using a large number of fluorochrome-labeled antibodies is hampered by the fact that suboptimal fluorochromes must be used, and that high and low cell density markers must be chosen with care. This is further complicated when the cell markers of interest are cytokines, transcription factors, surface markers, and/or phosphorylated proteins, each potentially requiring a specialized buffer system for optimal detection of the antibody of interest. This chapter focuses on optimizing flow cytometry staining methods for simultaneous detection of surface markers, transcription factors, secreted cytokines, and phosphorylated antibodies in a single stain on CD4+ human Th2 cells.

Automated High-Content Screening of γ-H2AX Expression in HeLa Cells.

Due to their inherent nature, DNA strands can be easily broken by various environmental factors including chemical agents and ionizing radiation. Unrepaired DNA double-stranded breaks (DSBs) may result in genetic instability and have a strong negative impact on the integrity of the genome. It has been found that DSBs are always followed by phosphorylation of histone protein H2AX, a member of the H2A family, and immunocytochemical detection of phosphorylated H2AX (referred to as γ-H2AX) is one of the frequently used techniques for assessing DNA damage. Usually such an assessment is done manually under the microscope which is not practical for analyzing large numbers of cells and prevents researchers from rapid and unbiased testing of novel drug compounds. To solve this problem we attempted to do automated assessment of DSBs by using a High-Content Screening (HCS) platform. As a result of this effort, we developed an easy to run HCS protocol for accurate analysis of DSBs in HeLa cells treated with camptothecin as a model. By varying the time of camptothecin treatment and its concentration we were able to study the dynamics of DSBs and perform a statistical analysis.Results of our study indicate that DSBs can be investigated using a HCS platform that enable the analysis of large numbers of experimental data points in a fast and a highly accurate manner. The protocol presented in this chapter can be easily adapted for screening libraries containing substantial numbers of chemical compounds for their efficiency to induce or/and repair DNA breaks.

Immunohistochemistry with the anti-BRAF V600E (VE1) antibody: impact of pre-analytical conditions and concordance with DNA sequencing in colorectal and papillary thyroid carcinoma.

The most common of all activating BRAF mutations (T1799A) leads to a substitution of valine (V) to glutamic acid (E) at the position 600 of the amino acid sequence. The major goal of this study was to compare detection of the BRAF V600E mutation by DNA sequencing with immunohistochemistry (IHC) using the anti-BRAF V600E (VE1) antibody. Archival formalin fixed, paraffin embedded tissues from 352 patients with colon adenocarcinoma (n = 279) and papillary thyroid carcinoma (n = 73) were evaluated for the BRAF V600E mutation by sequencing and IHC. The discordant cases were re-evaluated by repeat IHC, SNaPshot and next-generation sequencing (NGS). Furthermore, the effect of pre-analytical variables on the utility of this antibody was evaluated in two xenograft mouse models.After resolving 15 initially discordant cases, 212 cases were negative for the BRAF V600E mutation by IHC. Of these, 210 cases (99.1%) were also negative by sequencing and two cases (0.9%) remained discordant. Of the 140 cases that were IHC positive for BRAF V600E, 138 cases were confirmed by sequencing (98.6%) and two cases remained discordant (1.4%). Overall, the negative predictive value was 99.1%, positive predictive value 98.6%, sensitivity 98.6%, specificity 99.1% and overall percentage agreement 98.9% (348/352 cases). Tissue fixation studies indicated that tissues should be fixed for 12-24 h within 2 h of tissue collection with 10% neutral buffered formalin.