The long term aims of the parent grant (Al 31268) are: a) To determine the generality of antibody-mediated catalysis; b) To define the role of antibody catalysis in autoimmunity and immunological defence; c) To identify natural antigens capable of eliciting catalytic antibody formation; d) To determine the structural features of antibodies underlying their binding and hydrolytic activities; and, e) To isolate catalytic antibodies with potential therapeutic or investigative utility by screening of the immune repertoire.
The specific aims of this FIRCA collaborative proposal are: l. To investigate (i) the frequency of hydrolytic anti-DNA antibodies and anti- VIP antibodies in patients with systemic lupus erythematosus (SLE) and asthma, respectively, and control healthy subjects and (ii) the kinetics and specificity of antibody catalyzed VIP and DNA hydrolysis; 2. To determine the contribution of antibody subunits in DNA hydrolysis and binding; and 3. To clone and express the cDNA for hydrolyzing antibodies using phage display library techniques. The experimental design is: a) Antibody purification from blood of healthy individuals, asthma patients and SLE patients; b) Preparation of radioactive DNA and VIP probes; c) Radioassay of DNA binding and hydrolysis by antibodies; d) Comparison of these activities in healthy and disease groups; e) Characterization of antibody kinetic properties and specificity using denatured DNA, native DNA and short oligonucleotides of defined length and sequence; f) Purification of antibody Fab fragments and light chains; g) Comparison of the Km, turnover number and specificity of intact antibodies and isolated antibody subunits; h) Cloning and expression of recombinant single chain Fab by selection from phage display libraries.
| Demin, A V; Kolesnikov, A V; Olfer'ev, M A et al. (1998) [Expression of the catalytically active light chain of the antibody against vasoactive intestinal peptide] Dokl Akad Nauk 360:547-9 |
| Gabibov, A G; Makarevitch, O (1995) DNA hydrolysis by antibodies. Methods Mol Biol 51:223-35 |
| Schourov, D V; Gololobov, G V; Makarevich, O I et al. (1995) DNA-hydrolyzing autoantibodies in autoimmune pathologies. Ann N Y Acad Sci 750:255-64 |
| Gololobov, G V; Chernova, E A; Schourov, D V et al. (1995) Cleavage of supercoiled plasmid DNA by autoantibody Fab fragment: application of the flow linear dichroism technique. Proc Natl Acad Sci U S A 92:254-7 |
| Gabibov, A G; Gololobov, G V; Makarevich, O I et al. (1994) DNA-hydrolyzing autoantibodies. Appl Biochem Biotechnol 47:293-302;discussion 303 |
