| Molecular recognition is an integral part of several biological processes and systems and occurs between cells, proteins and nucleic acids. The molecular recognition between leukocytes and endothelial cells direct the migration of leukocytes from the blood to sites of pathological inflammation. Molecular recognition between proteins and nucleic acids are utilized in various molecular biology techniques such as enzyme-linked immunosorbent assays (ELISA) and quantitative real time polymerase chain reactions (q-PCRs).;Leukocyte - endothelial interactions play a key role in pathological inflammation and blocking such interactions would help in preventing the advancement of the disease. Several studies have shown that E-selectin, expressed on inflamed endothelium, and proteins and/or lipids decorated with sialyl Lewis x (sLe x), present on the surface of leukocytes, play a major role in the initial leukocyte -- endothelial interactions. It is reported that the monoclonal antibody (mAb) HECA-452 recognizes sLex. This observation motivated us to determine if the mAb HECA-452 blocks sLex mediated adhesion to endothelial expressed E-selectin. The results of this study showed that HECA-452 recognizes sLex and does not block sLe x mediated adhesion to endothelial expressed E-selectin. HECA-452 recognition of sLex does not depend on the fucose moiety to the extent required for E-selectin recognition. Combined, this study suggests that the HECA-452 reactive epitope(s) and the E-selectin binding epitope(s) of sLex are distinct.;Atherosclerosis is a complex disease process and it has been shown that Wnt5a and VCAM-1 are overexpressed at sites of atherosclerosis. As a first step towards the development of a diagnostic assay based on Wnt5a, we developed a sandwich ELISA (a protein recognition assay) for the detection of Wnt5a in buffers. The results of this study demonstrate that rabbit anti-human Wnt5a and goat anti-mouse Wnt5a are suitable antibodies for developing a Wnt5a sandwich ELISA. Furthermore, we found that the rabbit anti-human Wnt5a has to be the capture antibody. Finally, the results of this study revealed that adding PEG during both the binding of rm-Wnt5a and detection antibody stages of the assay yielded the maximum signal for rm-Wnt5a.;In the last study, we asked if the serum level of VCAM-1 and Wnt5a mRNA transcripts correlate with the extent of atherosclerosis using the ApoE -/- mouse model of atherosclerosis and q-PCR (a nucleic acid recognition assay). In this preliminary study, we found that the size and number of atherosclerotic plaques in ApoE-/- mice increases with age when kept on a high fat diet. Our preliminary q-PCR results revealed that comparison of the q-PCR data derived from different treatment groups is complex. Determination of an effective technique to compare the q-PCR results is needed to evaluate the relative expression of VCAM-1 and Wnt5a among the treatment groups.;Collectively, in this dissertation, the molecular recognition between cells, proteins and nucleic acids was applied to studies directly related to the development of novel therapeutics and diagnostics for pathological inflammation. |
