Simian virus 40 large tumor antigen (SV40 T antigen) untwists DNA at the SV40 replication origin. In the presence of ATP, T antigen shifted the average linking number of an SV40 origin-containing plasmid topoisomer distribution. The loss of up to two helical turns was detected. The reaction required the presence of the 64-base pair core origin of replication containing T antigen DNA binding sit...

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and (19)F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or indiv...

Antibodies are capable of specifically recognizing and binding antigens. Identification of the antigen-binding site, commonly dubbed paratope, is of high importance both for medical and biological applications. To date, the identification of antigen-binding regions (ABRs) relies on tools for the identification of complementarity-determining regions (CDRs). However, we have shown that up to 22% ...

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain po...

The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect...

We describe a highly sensitive heterogeneous enzyme-linked immunoassay in which digoxin is used as the model analyte. An excess of enzyme-labeled monovalent antibody is incubated with sample containing the analyte such that all analyte is rapidly and quantitatively bound. Excess antibody that does not acquire a antigen in its binding site is rapidly removed from the mixture by passage through a...

The orientation of the biological molecule immobilized on a solid surface has been critical in development of various applications. In this study, orientation of antibody was retained by protecting the antigen-binding site of the antibody prior to immobilization to -functionalized mixed self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 1-heptanethiol. More importantly, the numbe...

Epitope-G1 of bovine ephemeral fever virus (BEFV) G glycoprotein has been genetically and antigenically conserved among various isolates of BEFV and only reacts with anti-BEFV neutralising antibodies. Therefore, it is a candidate antigen for development of the enzyme linked immunosorbent assay (ELISA) for serological identification bovine ephemeral fever (BEF)-infected animals. The aim of this ...

Using a modified "two-site" immunoradiometric assay, termed an "interference assay," we have demonstrated the occurrence of non-analyte antibody-binding substances in approximately 40% of serum samples. These substances multivalently bind antibodies from any of several species at a site other than the antigen-binding site. Using a two-site immunoradiometric assay for human choriogonadotropin, w...

The success of antibody-based drugs has led to an increased demand for predictive computational tools to assist antibody engineering efforts surrounding the six hypervariable loop regions making up the antigen binding site. Accurate computational modeling of isolated protein loop regions can be quite difficult; consequently, modeling an antigen binding site that includes six loops is particular...