An assessment of histone-modification antibody quality.
ABSTRACT: We have tested the specificity and utility of more than 200 antibodies raised against 57 different histone modifications in Drosophila melanogaster, Caenorhabditis elegans and human cells. Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot. Among specific antibodies, more than 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use, and we provide a website for posting new test results (http://compbio.med.harvard.edu/antibodies/).
Project description:Three monoclonal antibody subclasses (IgG1, IgG2a, and IgM) were raised to the phosphoprotein B2 (Mr 68000, pI6.5-8.2) which has been shown previously to be associated with the nucleosomes of rat liver nuclei. These antibodies do not show any significant cross reactivity with CM-cellulose 'unbound' non-histone chromosomal proteins, bovine serum albumin or histones. Further verification of the specificity of these antibodies to this phosphoprotein was carried out using both 'dot' blot and immunological transfer analysis ('Western blot'). The monoclonal antibodies (IgG1 and IgG2a) could also be used to semi-quantify the phosphoprotein B2 in rat liver nuclei. The high specificity and unlimited availability of this type of probe provides a means to study the role(s) of this phosphoprotein in the overall scheme of actively transcribed chromatin.
Project description:The present study was aimed to develop and evaluate dot-blot assays for rapid detection of staphylococcal enterotoxin-A (SEA) in food. Dot blots were developed in two formats, indirect and sandwich utilizing mouse monoclonal anti-SEA and rabbit polyclonal anti-SEA antibodies. In indirect dot-blot format, recombinant SEA was directly coated on NCM dot-blot strip and detection was carried out by anti-SEA antibodies. In sandwich dot-blot format, SEA was trapped between anti-SEA capture and detection antibodies. Both the dot-blot assays exhibited a sensitivity of ~48 ng ml<sup>-1</sup> when tested in different food matrices. The developed assays were highly specific as no cross-reactivity was detected with other classical staphylococcal enterotoxins, toxigenic bacteria and foodborne pathogens. Sensitivity and specificity of developed indirect and sandwich dot-blot assays with respect to PCR was found to be 100 and 99%, respectively. The results shows that the developed dot-blot assays can be used as rapid preliminary screening tests for detection of SEA in food or determining the toxigenic potential of staphylococci, especially in resource-limited settings.
Project description:Chromatin structure is greatly influenced by histone tail post-translational modifications (PTM), which also play a central role in epigenetic processes. Antibodies against modified histone tails are central research reagents in chromatin biology and molecular epigenetics. We applied Celluspots peptide arrays for the specificity analysis of 36 commercial antibodies from different suppliers which are directed towards modified histone tails. The arrays contained 384 peptides from 8 different regions of the N-terminal tails of histones, viz. H3 1-19, 7-26, 16-35 and 26-45, H4 1-19 and 11-30, H2A 1-19 and H2B 1-19, featuring 59 post-translational modifications in many different combinations. Using various controls we document the reliability of the method. Our analysis revealed previously undocumented details in the specificity profile. Most of the antibodies bound well to the PTM they have been raised for, but some failed. In addition some antibodies showed high cross-reactivity and most antibodies were inhibited by specific additional PTMs close to the primary one. Furthermore, specificity profiles for antibodies directed towards the same modification sometimes were very different. The specificity of antibodies used in epigenetic research is an important issue. We provide a catalog of antibody specificity profiles for 36 widely used commercial histone tail PTM antibodies. Better knowledge about the specificity profiles of antibodies will enable researchers to implement necessary control experiments in biological studies and allow more reliable interpretation of biological experiments using these antibodies.
Project description:BACKGROUND:Bovine ephemeral fever virus (BEFV) causes fever and muscle stiffness in cattle, resulting in negative economic impact for cattle and dairy farms. During the manufacturing process of inactivated vaccine for virus control, it is important to determine the virus titer, but traditional methods such as plaque assay and TCID50 assay require days of waiting time. We sought to develop a quick dot blot assay for BEFV titering. RESULTS:Three different kinds of BEFV antigens were prepared to raise primary antibodies for BEFV detection in dot blot assays: 1) purified BEFV particles, 2) Escherichia coli (E. coli)-expressed BEFV G1 region, and 3) E. coli-expressed BEFV N protein. Results showed that antibodies raised against purified BEFV particles can detect BEFV particles, but it also showed a high background level from the proteins of BHK-21 cells. Antibodies raised against E.coli-expressed BEFV G1 region could not detect BEFV particles in dot blot assays. Finally, antibodies raised against E.coli-expressed BEFV N protein detected BEFV particles with a high signal-to-noise ratio in dot blot assays. CONCLUSIONS:E.coli-expressed N protein is a suitable antigen for the production of antiserum that can detect BEFV particles with a high signal-to-noise ratio. A dot blot assay kit using this antiserum can be developed as a quick and economical way for BEFV titering.
Project description:DNA encoding the coat protein (CP) of an Egyptian isolate of tomato yellow leaf curl virus (TYLCV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) under the control of polyhedrin promoter. The generated recombinant baculovirus construct harboring the coat protein gene was characterized using PCR analysis. The recombinant coat protein expressed in infected insect cells was used as a coating antigen in an indirect Enzyme-linked immunosorbent assay (ELISA) and dot blot to test its utility for the detection of antibody generated against TYLCV virus particles. The results of ELISA and dot blot showed that the TYLCV-antibodies reacted positively with extracts of infected cells using the recombinant virus as a coating antigen with strong signals as well as the TYLCV infected tomato and beat plant extracts as positive samples. Scanning electron microscope examination showed that the expressed TYLCV coat protein was self-assembled into virus-like particles (VLPs) similar in size and morphology to TYLCV virus particles. These results concluded that, the expressed coat protein of TYLCV using baculovirus vector system is a reliable candidate for generation of anti-CP antibody for inexpensive detection of TYLCV-infected plants using indirect CP-ELISA or dot blot with high specificity.
Project description:BACKGROUND: RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. METHODS: We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. RESULTS: The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. CONCLUSION: We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3 expression in normal and cancerous human breast tissue at both the mRNA and protein levels.
Project description:Mutations in Cu/Zn-superoxide dismutase (SOD1) gene are linked to 10-20% of familial amyotrophic lateral sclerosis (fALS) cases. The mutations cause misfolding and self-assembly of SOD1 into toxic oligomers and aggregates, resulting in motor neuron degeneration. The molecular mechanisms underlying SOD1 aggregation and toxicity are unclear. Characterization of misfolded SOD1 is particularly challenging because of its metastable nature. Antibodies against misfolded SOD1 are useful tools for this purpose, provided their specificity and selectivity are well-characterized. Here, we characterized three recently introduced antimisfolded SOD1 antibodies and compared them with two commercial, antimisfolded SOD1 antibodies raised against the fALS-linked variant G93A-SOD1. As controls, we compared the reactivity of these antibodies to two polyclonal anti-SOD1 antibodies expected to be insensitive to misfolding. We asked to what extent the antibodies could distinguish between WT and variant SOD1 and between native and misfolded conformations. WT, G93A-SOD1, or E100K-SOD1 were incubated under aggregation-promoting conditions and monitored using thioflavin-T fluorescence, electron microscopy, and dot blots. WT and G93A-SOD1 also were analyzed using native-PAGE/Western blot. The new antimisfolded SOD1 and the commercial antibody B8H10 showed variable reactivity using dot blots but generally showed maximum reactivity at the time misfolded SOD1 oligomers were expected to be most abundant. In contrast, only B8H10 and the control antibodies were reactive in Western blots. Unexpectedly, the polyclonal antibodies showed strong preference for the misfolded form of G93A-SOD1 in dot blots. Surprisingly, antimisfolded SOD1 antibody C4F6 was specific for the apo form of G93A-SOD1 but insensitive to misfolding. Antibody 10C12 showed preference for early misfolded structures, whereas 3H1 bound preferentially to late structures. These new antibodies allow distinction between putative early- and late-forming prefibrillar SOD1 oligomers.
Project description:The development of a sensitive and rapid diagnostic test is needed for early detection of avian influenza (AI) H7 subtype. In this study, novel monoclonal antibodies (mAbs) against influenza A H7N9 recombinant hemagglutinin (rHA)1 were developed and applied to a Europium nanoparticle-based rapid fluorescent immunochromatographic strip test (FICT) to improve the sensitivity of the rapid diagnostic system. Two antibodies (2F4 and 6D7) exhibited H7 subtype specificity in a dot-FICT assay by optimization of the conjugate and the pH of the lysis buffer. The subtype specificity was confirmed by an immunofluorescence assay and Western blot analysis. The limit of detection of the FICT employing novel mAbs 31 ng/mL for H7N9 rHA1 and 40 hemagglutination units/mL for H7 subtype virus. Sensitivity was improved 25-fold using Europium as confirmed by comparison of colloidal gold-based rapid diagnostic kit using the 2F4 and 6D7 mAbs.
Project description:BACKGROUND:Histone posttranslational modifications (PTMs) represent a focal point of chromatin regulation. The genome-wide and locus-specific distribution and the presence of distinct histone PTMs is most commonly examined with the application of histone PTM-specific antibodies. In spite of their central role in chromatin research, polyclonal antibodies suffer from disadvantages like batch-to-batch variability and insufficient documentation of their quality and specificity. RESULTS:To mitigate some of the pitfalls of using polyclonal antibodies against H3K4me3, we successfully validated the application of a recombinant TAF3 PHD domain as anti-H3K4me3 affinity reagent in peptide array, western blot and ChIP-like experiments coupled with qPCR and deep sequencing. CONCLUSIONS:The successful addition of the TAF3 PHD domain to the growing catalog of recombinant affinity reagents for histone PTMs could help to improve the reproducibility, interpretation and cross-laboratory validation of chromatin data.
Project description:Rice tungro disease (RTD) is one of the most destructive diseases of rice in South and Southeast Asia. RTD is routinely detected based on visual observation of the plant. However, it is not always easy to identify the disease in the field as it is often confused with other diseases or physiological disorders. Here we report the development of two serological based assays for ease of detection of RTD. In this study we had developed and optimized an indirect ELISA and dot-blot assay for detection of RTD. The efficiency of both assays was evaluated by comparing the specificity and sensitivity of the assays to PCR assay using established primer sets. The indirect ELISA showed 97.5% and 96.6%, while the dot-blot assay showed 97.5% and 86.4% sensitivity and specificity, respectively, when compared to established PCR method. The high sensitivity and specificity of the two assays merit the use of both assays as alternative methods to diagnose RTD. Furthermore, the dot-blot assay is a simple, robust, and rapid diagnostic assay that is suitable for field test for it does not require any specialized equipment. This is a great advantage for diagnosing RTD in paddy fields, especially in the rural areas.