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By Veena Venkatachalam • Neurons – cells that make up our nervous systems • have processes that transmit electrical impulses (action potentials) • this is how your brain communicates with the rest of your body • neurodegenerative diseases cause these neurons to die • basic fibroblast growth factor (bFGF) promotes neuron survival • bFGF – growth factor that stimulates elongation and branching of neuronal processes • Why don’t we use bFGF to treat patients with Alzheimer’s or Parkinson’s disease? It degrades too rapidly! • Solution – find a stable substance that has bFGF-like effects • Antibodies – Y-shaped proteins that are produced by cells of the immune system in response to a stimulus; protect us by neutralizing toxins • four chains – two heavy chains and two light chains • each chain has one variable region; variable regions differentiate antibodies from one other and determine specificity • We have hypothesized, based on past observations, that serum from several patients contains an antibody that has effects similar to those of bFGF Complementary Complementary bFGF (neurotrophic growth factor) Idiotype (antibody complementary to bFGF) Anti-idiotype (bFGFlike antibody; similar antigen binding site) • We used three different methods to purify the patient serum: Protein A beads: bind IgGs Protein A beads: pour serum over column and elute with citric acid; result => only IgGs Hydroxyapatite gel Hydroxyapatite (HA) column: pour protein A eluate over column, elute w/ diff. molarities of sodium phosphate Immunoaffinity column Immunoaffinity column: has anti-bFGF antibodies bound to the protein A beads; thus, bFGF like antibodies bind very strongly. Elute w/ diff. pH • Bioactivity assays were used to determine the fraction that we wanted to purify further pH fractions from IA column: bioassay bioactivity (endothelial cell assay) protein conc 0.1 0.09 percent of control 150.00% 0.08 0.07 140.00% 0.06 130.00% 0.05 0.04 120.00% 0.03 0.02 110.00% 0.01 100.00% 0 Control pH 3.3 pH 3.0 pH 2.8 pH 2.65 pH 2.3 protein concentration (m g/m L) 160.00% I’d like to extend my deepest appreciation to Dr. Zimering for his guidance. I would also like to thank Robert Donnelly for his help in running mass spectroscopy and amino acid sequencing our samples.