Characterization of an IDH1 R132H Rabbit Monoclonal Antibody, MRQ-67, and Its Applications in the Identification of Diffuse Gliomas.

The current diagnosis of diffuse glioma involves isocitrate dehydrogenase (IDH) mutation testing. Most IDH mutant gliomas carry a G-to-A mutation at IDH1 position 395, resulting in the R132H mutant. R132H immunohistochemistry (IHC), therefore, is used to screen for the IDH1 mutation. In this study, the performance of MRQ-67, a recently generated IDH1 R132H antibody, was characterized in comparison with H09, a frequently used clone. Selective binding was demonstrated by an enzyme-linked immunosorbent assay for MRQ-67 to the R132H mutant, with an affinity higher than that for H09. By Western and dot immunoassays, MRQ-67 was found to bind specifically to the IDH1 R1322H, with a higher capacity than H09. IHC testing with MRQ-67 demonstrated a positive signal in most diffuse astrocytomas (16/22), oligodendrogliomas (9/15), and secondary glioblastomas tested (3/3), but not in primary glioblastomas (0/24). While both clones demonstrated a positive signal with similar patterns and equivalent intensities, H09 exhibited a background stain more frequently. DNA sequencing on 18 samples showed the R132H mutation in all IHC positive cases (5/5), but not in negative cases (0/13). These results demonstrate that MRQ-67 is a high-affinity antibody suitable for specific detection of the IDH1 R132H mutant by IHC and with less background as compared with H09.

Characterization of polyclonal antibodies to Herpes Simplex Virus types 1 and 2.

Infections with herpes simplex virus (HSV) types 1 and 2 have been linked to oral, facial, genital lesions, as well as some visceral organ changes in patients under immunosuppressed conditions. Immunohistochemistry (IHC) with HSV antibodies is used for identification of the viruses in tissue samples. In this study, two polyclonal antibodies, prepared separately with HSV-1 and HSV-2 immunogens, were characterized in comparison to a monoclonal antibody to HSV-1 (10A3). The polyclonal anti-HSV-1 and monoclonal antibody 10A3 were shown to be reactive to viral proteins of both HSV-1 and HSV-2 on Western blots, while the polyclonal anti-HSV-2 was reactive to HSV-2 proteins, but not to those of HSV-1. Cross-reactivity was not observed to proteins of six other frequently encountered herpes viruses. IHC characterization was performed on 29 cases of HSV-infected tissue samples, 61 samples infected with other herpes viruses and 35 samples without known infection. By IHC, the polyclonal anti-HSV-1 and a monoclonal antibody 10A3 exhibited a signal, mainly in a nuclear pattern, in all of the HSV-infected samples and not in other tissue types. A positive signal, mainly in the cytoplasm, was identified with the polyclonal anti-HSV-2 in 21 of the 29 HSV-infected samples. Genotyping analysis was successful in 14 of the HSV-infected samples, with IHC HSV-2 positivity correlative to the HSV-2 genotype. The results demonstrate that these antibodies are useful tools for identification of HSV-1 and HSV-2, and their combinatorial application may help to distinguish between these two types of infection.

Generation and Performance of R132H Mutant IDH1 Rabbit Monoclonal Antibody.

Isocitrate dehydrogenase 1 (IDH1) gene mutations have been observed in a majority of diffuse astrocytomas, oligodendrogliomas, and secondary glioblastomas, and the mutant IDH1 R132H is detectable in most of these lesions. By specifically targeting the R132H mutation through B-cell cloning, a novel rabbit monoclonal antibody, MRQ-67, was produced that can recognize mutant IDH1 R132H and does not react with the wild type protein as demonstrated by Enzyme-linked immunosorbent assay (ELISA) and Western blotting. Through immunohistochemistry, the antibody is able to highlight neoplastic cells in glioma tissue specimens, and can be used as a tool in glioma subtyping. Immunohistochemistry (IHC) detection of IDH1 mutant protein may also be used to visualize single infiltrating tumor cells in surrounding brain tissue with an otherwise normal appearance.

Same-Single-Cell Analysis of Pacemaker-Specific Markers in Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Subtypes Classified by Electrophysiology.

Insights into the expression of pacemaker-specific markers in human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker-, atria-, and ventricular-like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles. Traditional acquisition of action potentials using patch-clamp recordings renders the cells unviable for subsequent analysis. We circumvented these issues by acquiring the action potential profile of a single cell optically followed by assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the first time revealed expression of proposed pacemaker-specific markers-hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet (Isl)1-at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expression was found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- and ventricular-like subtypes but its downregulation over time in all subtypes diminished the differences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statistically different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentially expressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes, these markers alone are insufficient in identifying hiPSC-derived pacemaker-like cardiomyocytes. Stem Cells 2016;34:2670-2680.