Download Antibodyexperiment

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.