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

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

Antibodies to the Mr 64,000 (64K) protein in islet cell antibody positive non-diabetic individuals indicate high risk for impaired Beta-cell function

A prospective study of a normal childhood population identified 44 islet cell antibody positive individuals. These subjects were typed for HLA DR and DQ alleles and investigated for the presence of antibodies to the Mr 64,000 (64K) islet cell antigen, complement-fixing islet cell antibodies and radiobinding insulin autoantibodies to determine their potency in detecting subjects with impaired Beta-cell function. At initial testing 64K antibodies were found in six of 44 islet cell antibody positive subjects (13.6%). The same sera were also positive for complement-fixing islet cell antibodies and five of them had insulin autoantibodies. During the follow-up at 18 months, islet cell antibodies remained detectable in 50% of the subjects studied. In all six cases who were originally positive, 64K antibodies were persistently detectable, whereas complement-fixing islet cell antibodies became negative in two of six and insulin autoantibodies in one of five individuals. HLA DR4 (p < 0.005) and absence of asparic acid (Asp) at position 57 of the HLA DQ chain (p < 0.05) were significantly increased in subjects with 64K antibodies compared with control subjects. Of 40 individuals tested in the intravenous glucose tolerance test, three had a first phase insulin response below the first percentile of normal control subjects. Two children developed Type 1 (insulin-dependent) diabetes mellitus after 18 and 26 months, respectively. Each of these subjects was non-Asp homozygous and had persistent islet cell and 64K antibodies. We conclude that 64K antibodies, complement-fixing islet cell antibodies and insulin autoantibodies represent sensitive serological markers in assessing high risk for a progression to Type 1 diabetes in islet cell antibody positive non-diabetic individuals

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

Factors influencing transfusion-associated HLA sensitization in patients bridged to heart transplantation using ventricular assist device.

BackgroundBridging heart failure patients with mechanical ventricular assist devices (VAD) enables access to transplantation. However, VAD is associated with increased risk for anti-HLA antibodies associated with rejection of subsequent allografts. Factors determining alloantibody formation in these patients remain undefined.MethodsWe performed a single-center retrospective cohort study of 164 patients undergoing heart transplantation from 2014 to 2017. Medical records including use of VAD, transfused blood products, anti-HLA antibody testing, crossmatch, and time to transplant were evaluated.ResultsPatients received an average of 13.8 red blood cell and 1.9 single-donor platelet units associated with VAD. There was a 28.7% increase in the incidence of anti-HLA antibodies after VAD. Development of anti-HLA antibodies did not correlate with volume or type of blood products, but with pre-VAD HLA sensitization status; relative risk of new alloantibodies in patients with pre-VAD antibodies was 3.5-fold higher than those without prior antibodies (P = .008). Development of new anti-HLA antibodies was associated with an increased time to transplant (169 vs 330 days, P = .013).ConclusionsOur findings indicate that the presence of anti-HLA antibodies pre-VAD was the most significant risk factor for developing additional antibodies post-VAD, suggesting that a subset of patients may be predisposed to alloantibody formation

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