The long term goals of our research programs are: (i) to develop an understanding of the basis of catalysis by antibodies, (ii) to determine the physiological or pathophysiological functions of peptidolytic antibodies, and (iii) to develop catalytic anti-peptide antibodies for use as research and therapeutic tools. Specific endopeptidase activity of human autoantibodies to VIP has been reported. Recent findings suggest that immunization with VIP elicits catalytic antibodies and that dissociated light chains from VIP antibodies display increased catalytic activity. These observations raise new questions concerning the antibody catalytic function and its relation to the binding function. This proposal will address these questions through investigation of available murine monoclonal antibodies elicited against VIP, examination of hybrid antibody oligomers and, determination of catalytic and binding antibody responses to VIP and other polypeptides. The specific objectives of the proposed studies are: 1. To characterize the catalytic and binding properties of murine monoclonal antibodies to VIP and their component L- and H-chains (turnover number, Km, Kd, peptide bond specificity, epitope specificity and susceptibility to product inhibition); 2. To clone and express the cDNA for the H- and L-chains; 3. To prepare hybrid antibodies with catalytic activity. 4. To investigate the catalytic antibody response to peptides without homology to VIP and peptides designed to feature structural motifs found in VIP. The antibodies will be screened by direct methods for detection of peptidase activity using antigen or antigen analogs as substrate. Various chromatographic and electrophoretic methods will be applied. Products are isolated and identified by peptide sequencing and FAB-mass spectrometry. Standard DNA methods will be used to obtain cDNA from hybridoma cells, amplify the cDNA by PCR, and clone the cDNA.
| Nishiyama, Yasuhiro; Planque, Stephanie; Mitsuda, Yukie et al. (2009) Toward effective HIV vaccination: induction of binary epitope reactive antibodies with broad HIV neutralizing activity. J Biol Chem 284:30627-42 |
