This application is a competing renewal to continue research to develop a sensitive and specific ELISA-based test to measure hemoglobin-acetaldehyde adducts (Hb-AAs) and to assess the relationship of this test to the 1,3-cyclohexanedione (CHD) fluorogenic HPLC assay. In the preceding granting period, we have developed three first-generation antibodies (Abs), i.e. anti-keyhole limpet hemocyanin-AA IgG (anti-KLH-AA), anti-GK-ll-peptide-AA IgG (G56 to K66 peptide in beta-chain of HbA), and anti-VK-8-peptide-AA IgG (VI to K8 peptide in beta-chain of HbS). The sensitivity of anti-KLH-AA IgG was the highest, i.e. 78%. In this new proposal, we hypothesize that (1) abnormally elevated Hb-AA is found in vivo in alcoholic patients, and highly specific monoclonal antibodies (Mabs) can be raised against Hb-AA after its isolation and purification; (2) these Mabs will react with irreversibly bound acetaldehyde as epitopes and not with reversibly bound AAs; (3) polyclonal Abs prepared by the """"""""multiple antigen-peptide (MAP) system"""""""" are better antibodies (Abs) than polyclonal Abs prepared by conjugating Hb peptides to carrier proteins such as KLH; and (4) HbAA measurement can be developed to become a very useful diagnostic marker of alcohol abuse. In addition to testing these hypothesis, two other specific aims will be pursued: (1) To elucidate the location and structure of AA epitopes in Hb-AA by coupling protease digestion to mass spectroscopic studies and to amino acid sequencing. (2) To identify by CC/MS the aldehydes reversibly bound to red cells which purportedly can be detected by CHD fluorogenic HPLC. Our laboratory (including our Biotechnology Center) is well equipped to synthesize appropriately selected Hb peptide monomers and MAP-peptides and their AAs, to do protease digestion and HPLC separation, to prepare affinity resins, polyclonal and monoclonal Abs, to do protein sequencing and to perform various HPLC and immunologic assays. Collaborations will be needed for mass spectroscopic analysis of CHD-acetaldehyde adducts, mass spectroscopic analysis of Hb and peptides to localize the AA in Hb-AA and collection of larger number of blood samples. The latter will supplement blood samples that we can collect within hospitals at the Indiana University Medical Center.
