Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq.

BackgroundThe robustness of ChIP-seq datasets is highly dependent upon the antibodies used. Currently, polyclonal antibodies are the standard despite several limitations: They are non-renewable, vary in performance between lots and need to be validated with each new lot. In contrast, monoclonal antibody lots are renewable and provide consistent performance. To increase ChIP-seq standardization, we investigated whether monoclonal antibodies could replace polyclonal antibodies. We compared monoclonal antibodies that target five key histone modifications (H3K4me1, H3K4me3, H3K9me3, H3K27ac and H3K27me3) to their polyclonal counterparts in both human and mouse cells.ResultsOverall performance was highly similar for four monoclonal/polyclonal pairs, including when we used two distinct lots of the same monoclonal antibody. In contrast, the binding patterns for H3K27ac differed substantially between polyclonal and monoclonal antibodies. However, this was most likely due to the distinct immunogen used rather than the clonality of the antibody.ConclusionsAltogether, we found that monoclonal antibodies as a class perform equivalently to polyclonal antibodies for the detection of histone post-translational modifications in both human and mouse. Accordingly, we recommend the use of monoclonal antibodies in ChIP-seq experiments

Engineering Synthetic Antibody by Expanded Genetic Code

Antibodies are extensively used in research for diagnostic and therapeutic purposes because of their unrivaled specificity and biomarker binding strengths.1 Currently, monoclonal antibodies are most commonly used because of their production consistency and purity.1 However, there are significant ethical and economic challenges associated with producing monoclonal antibodies.1 Synthetic antibodies provide a promising alternative to monoclonal antibodies in both clinical and research applications.2 Our proposed synthetic antibody system incorporates 3,4-dihydroxy-l-phenylalanine (L-DOPA), an unnatural amino acid used to increase binding affinity, into a peptide sequence specific for the prostate specific antigen (PSA), a biomarker for prostate cancer. This addition is predicted to give the synthetic antibody binding affinity and PSA specificity comparable to existing monoclonal antibodies while avoiding their drawbacks.3 If successful, our system would replace monoclonal antibodies for PSA detection as well as be a promising model for developing countless other synthetic antibodies

Differential binding patterns of anti-sulfatide antibodies to glial membranes

Sulfatide is a major glycosphingolipid in myelin and a target for autoantibodies in autoimmune neuropathies. However neuropathy disease models have not been widely established, in part because currently available monoclonal antibodies to sulfatide may not represent the diversity of anti-sulfatide antibody binding patterns found in neuropathy patients. We sought to address this issue by generating and characterising a panel of new anti-sulfatide monoclonal antibodies. These antibodies have sulfatide reactivity distinct from existing antibodies in assays and in binding to peripheral nerve tissues and can be used to provide insights into the pathophysiological roles of anti-sulfatide antibodies in demyelinating neuropathies

Characterization of monoclonal and polyclonal antibodies to bovine enteric coronavirus: Establishment of an efficient ELISA for antigen detection in feces

Monoclonal antibodies to bovine enteric coronavirus (BEC) were produced. Additionally, polyclonal antibodies were made in rabbits and guinea pigs and extracted from the yolk of immunized hens. The antibodies were characterized by neutralization test, hemagglutination inhibition test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Neutralizing antibody titers of polyclonal antisera ranged from 1:1280 to 1:40 000. Only one out of 908 hybridoma colonies tested secreted antibodies with neutralizing activity. By ELISA, polyclonal sera exhibited high background reactions that could be significantly reduced by treatment with kaolin in the case of rabbit sera. Attempts to establish an ELISA for BEC antigen detection based on polyclonal sera failed due to low sensitivity and specificity. Optimal results were achieved when a mixture of two monoclonal antibodies was coated onto microplates for antigen capture, while rabbit hyperimmune serum served as detecting antibodies in an indirect assay. The combination of the two monoclonal antibodies did not increase sensitivity synergistically, but in a compensatory fashion, probably because of epitope differences between BEC field strain

Monoclonal Antibodies against the Drosophila Nervous System

A panel of 148 monoclonal antibodies directed against Drosophila neural antigens has been prepared by using mice immunized with homogenates of Drosophila tissue. Antibodies were screened immunohistochemically on cryostat sections of fly heads. A large diversity of staining patterns was observed. Some antigens were broadly distributed among tissues; others were highly specific to nerve fibers, neuropil, muscle, the tracheal system, cell nuclei, photoreceptors, or other structures. The antigens for many of the antibodies have been identified on immunoblots. Monoclonal antibodies that identify specific molecules within the nervous system should prove useful in the study of the molecular genetics of neural development

Clathrin structure characterized with monoclonal antibodies. I. Analysis of multiple antigenic sites.

Three monoclonal antibodies that react with previously undefined antigenic determinants on the clathrin molecule have been produced and characterized. They were isolated from a fusion between myeloma cells and popliteal lymphocytes from SJL mice that had received footpad injections of human brain clathrin. This protocol was chosen to favor the production of antibodies to poorly immunogenic proteins and thereby increase the repertoire of anti-clathrin monoclonal antibodies. One antibody (X16) reacts preferentially with the heavier of the two clathrin light chains (LCa) when it is not associated with heavy chain. This specificity is different from that of the anti-LCa antibody, CVC.6, which has preferential reactivity with heavy chain-associated LCa. In addition, X16 and CVC.6 bound simultaneously to LCa, confirming that they react with different sites. The other two antibodies produced, X19 and X22, react with two different determinants on the clathrin heavy chain, based on immunoprecipitation, Western blot, and binding studies. Competitive binding studies with anti-clathrin monoclonal antibodies showed that they define a total of five distinct antigenic determinants on bovine clathrin

Rat Monoclonal Antibodies Specific for LST1 Proteins

The LST1 gene is located in the human MHC class III region and encodes transmembrane and soluble isoforms that have been suggested to play a role in the regulation of the immune response and are associated with inflammatory diseases such as rheumatoid arthritis. Here we describe the generation and characterization of the first monoclonal antibodies against LST1. Two hybridoma lines secreting monoclonal antibodies designated 7E2 and 8D12 were established. The 7E2 antibody detects recombinant and endogenous LST1 by Western blot analysis while 8D12 reacts with recombinant and endogenous LST1 in immunoprecipitation and flow cytometry procedures. The newly established antibodies were used to survey LST1 protein expression in human cell lines, which was found to be tightly regulated, allowing the expression of transmembrane isoforms but suppressing soluble isoforms

Dependence of endothelial cell growth on substrate-bound fibronectin

A better understanding of the mechanism of adhesion, spreading and proliferation of human endothelial cells (HEC) on polymeric surfaces may lead to the development of vascular prostheses which allow the formation of an endothelial lining on the luminal surface. In the present investigation the interaction of HEC with polyethylene precoated with monoclonal antibodies directed against HEC membrane antigens and against extracellular matrix compounds was studied. F(ab¿)2 fragments of a monoclonal antibody, directed against an endothelial cell membrane antigen, and F(ab')2 fragments of a monoclonal antibody, directed against cellular fibronectin, were also included in this study. Preadsorption of these antibodies and F(ab')2 fragments, including mixtures of antibodies and mixtures of F(ab')2 fragments, resulted in cell adhesion and spreading as well as moderate cell proliferation (or no proliferation) for several days. However, a good proliferation of HEC was only observed on polyethylene precoated with fibronectin or CLB-HEC-FN-140 (directed against fibronectin). These results strongly suggest that fibronectin, bound to a solid substrate, provides a biochemical signal necessary for the proliferation of HEC. The initial proliferation of HEC on other preadsorbed antibodies or F(ab')2 fragments may be explained by the fact that suspended HEC, used for cell seeding, still possess cell membrane-bound fibronectin