Browsing by Subject "ELISA"

Now showing 1 - 3 of 3
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    Analysis of the Mechanisms of Anti-DNA Antibody Binding to DNA
    (2020-05-08) Garza Reyna, Angel
    Antibodies to DNA (anti-DNA) are a canonical marker for systemic lupus erythematosus (SLE), a prototypic autoimmune disease that is most common in young women and has highly variable immunological and clinical manifestations. Anti-DNA antibodies play a key role in the pathogenesis of lupus as they can form pathogenic immune complexes that can ultimately lead to organ damage. Prior studies showed that the antibody’s charge may influence the formation of immune complexes; DNA is negatively charged because of the phosphodiester backbone and anti-DNA antibodies are rich in positively charged amino acids. To elucidate the mechanisms of anti-DNA antibodies, we wanted to further explore how charge affects antibody binding using as a model a monoclonal antibody. In this study, we tested whether the binding properties of Val-1205, an E310 monoclonal antibody (MAb), are mediated by electrostatic interactions, similar to those identified for serum as well as monoclonal anti-DNA antibodies. For this purpose, we took advantage of this monoclonal antibody that has been re-engineered to express more charge and higher binding avidity to determine how charge affects the association and dissociation of ICs. Enzyme-linked immunosorbent assays (ELISAs) were performed under distinct experimental environments to identify possible changes in antigen-antibody (Ag-Ab) interactions. We found that Val-1205 binds well to DNA in ELISAs and that changing buffer composition without altering pH did not significantly change binding activity. In addition, Val-1205 binding was unchanged by the number of times the antibody had been thawed. However, Val-1205’s binding activity was influenced by salt concentration. When compared to antibodies from SLE patient serum, the Val-1205 antibody had comparable dependence on ionic strength. These results are important since they demonstrate the exquisite dependence on the ionic strength of anti-DNA, reflecting the role of electrostatic interactions. Results presented herein provide new understandings of the mechanism of anti-DNA–DNA interaction and indicate that Val-1205 may be especially dependent on charge-charge interaction. These results also provide an important insight that Val-1205, despite its strong binding affinity, may represent anti-DNA antibodies that are primarily dependent on charge. The dependence on ionic interactions in Val-1205 may be utilized as steppingstones to better understand the immunopathogenesis of lupus.
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    Polymorphic variants of Fc receptors and antibodies derived from humans and rhesus macaques exhibit differential binding
    (2017-05-12) Penny, Caitlin
    Immune effector functions often depend on the fragment crystallizable (Fc) region of antibodies binding with Fc receptors (FcRs) on immune cells to trigger various responses. Polymorphisms in both Fc and FcR genes in humans and rhesus macaques have been demonstrated to alter the strength of this binding and consequently the immune response that is elicited. Rhesus macaques are often studied as an animal model for AIDS-like diseases, although he diversity of their FcRs has not yet been well characterized. Rhesus have more variation in their FcR genes, but less variation among IgG subclasses compared to humans. I hypothesize that the strength of signaling and subsequent immune responses caused by FcR-bearing cells will be regulated by the strength of Fc binding and the expression levels of FcRs on effector cells. To test this hypothesis, a more accurate genome map of human and rhesus macaques must be compiled, and methods developed to characterize interactions between polymorphic variants of FcRs and antibodies. I devised an ELISA protocol to test the hypothesis that known human and rhesus macaque FcR polymorphisms have differing binding affinities to antibody variants. My results suggest that ELISA assays can measure the strength of binding between variants of FcRs and antibodies to characterize interactions between these molecules. Future work should use similar ELISA techniques as well as immune complexes suspended in solution to distinguish the differing responses among a wider variety of both human and macaque polymorphisms within both FcR and antibody genes.
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    University of California San Francisco Lab Medicine Resident Critical Reviews - Disseminated Histoplasmosis and the Urinary Histoplasmosis Antigen
    (University of California San Francisco Lab Medicine Resident Critical Reviews, 1992-06-01) Gallagher, David
    Review article written for University of California San Francisco Laboratory Medicine "Critical reviews" in June 1992 by Dr David Gallagher. Covers background of disseminated histoplasmosis and diagnostic approach. Critical review of the urinary histoplasmosis antigen test available at the time.