Download SRTP presentation

Purification and Characterization
of Fibroblast Growth Factor-like
Antibodies
Introduction: bFGF
 Basic fibroblast growth factor (bFGF), is a single-chain polypeptide
composed of 146 amino acids.
 bFGF is a normal component of bone extracellular matrix as well as a
constituent of various normal and tumor tissues that does not normally
circulate in the serum of healthy non-pregnant adult humans.
 Previous studies have determined that bFGF has potent mitogenic and
neurotrophic functions, meaning it promotes the growth and
proliferation of certain types of cells (i.e. endothelial cells) as well as
survival and neurite outgrowth of brain neurons in vitro and in vivo.
(Zimering, M. B. et al. Circulating Fibroblast Growth Factor-Like Autoantibodies in Two Patients with Multiple
Endocrine Neoplasia Type 1 and Prolactinoma. Journal of Clinical Endocrinology and Metabolism 79.6 (1994) 15465152. )
Introduction: bFGF-like
Antibodies
 A bFGF-like substance has been identified by prior research in the
plasma of patients with various cancers of the endocrine system.
 This bFGF-like substance is an immunoglobulin G, which may have
been created as an antibody against anti-bFGF antibodies.
 The creation of such antiidiotype antibodies is not uncommon for the
immune system.
 In prior experiments, strong evidence has been gathered that the bFGFlike antibody mimics the growth-promoting effects of bFGF.
 An antibody remains in circulation much longer than bFGF and is
much more gradually degraded in plasma, thus making bFGF-like
antibody of considerable interest in procedures that require the growthpromoting effects of bFGF. (Zimering, M. B. et al. Circulating Fibroblast Growth Factor-Like
Autoantibodies in Two Patients with Multiple Endocrine Neoplasia Type 1 and Prolactinoma. Journal of Clinical
Endocrinology and Metabolism 79.6 (1994) 1546-5152. )
Primary Hypothesis
 Purification of bFGF-like antibody samples
enhances the biological activity (i.e. growthpromoting effects in endothelial cells) of the these
samples.
Purpose of Study
• To purify and characterize the biochemical
properties of the bFGF-like antibody.
• To answer questions about the activity of the
antibody at various dilutions so as to determine
peak biological activity.
• Ultimately, purification of the antibody is a means
at deriving its amino acid sequence, at which point
it might be synthesized in vitro.
Methods & Materials:
Methods of Purification
 Protein-A affinity chromatography: To separate the immunoglobulin G (IgG)
fraction from plasma, 0.5-mL aliquots of plasma were applied to a 1-mL column
of packed protein-A beads. The column was washed with 5 mL 10 mM Tris (flowthrough fraction), and then eluted stepwise with 5 x 1.0 mL .1 M (mol/L) citric
acid, pH 3.0. The second and third eluate fractions were pooled, and the rest were
discarded
 Hydroxyapatite chromatography: To further purify the IgG fraction from plasma,
0.5 mL aliquots of protein-A-eluted fractions were applied to a hydroxyapatite
column. The column was washed with 5 mL of 10 mM sodium phosphate (flowthrough fraction). Subsequently, the column was eluted with a linear gradient of
sodium phosphate (50-400 mM). The eluates were collected as separate 1 mL
fractions.
 Antihuman IgG affinity chromatography: To further purify the IgG fractions
obtained from hydroxyapatite chromatography, 0.5 mL aliquots of the 400 mM
hydroxyapatite fraction were applied to an immunoaffinity column. The column
was washed with 10 mL of 10mM Tris and eluted with a pH gradient of citric acid
(3.3 - 2.3 pH). The eluate were collected as separate 1.5 mL fractions of different
pH.
Methods & Materials:
Bioactivity Assays, Protein
Determination, & Mass Spec.
 Protein determination: Dye was added to minute amounts of the samples as well as
to protein standards of known concentrations. Upon comparing the absorbance
readings of the known and the unknown proteins, the protein concentrations of the
laboratory samples were determined.
 Mass spec: Modern time-of-flight mass spectrometry was used to determine the
precise molecular weights of multiple proteins from various laboratory samples (e.g.
hydroxyapatite fractions and protein-A eluate).
 Colorimetric assay: After being plated into 96-well plates along with test fractions,
endothelial cells were allowed to incubate for 4 days. Then, cell-associated
bioactivity was determined by colorimetry in an MR 700 plate reader (Dyna-Tech).
Growth-promoting activity is expressed as a percentage of the control cell number
for cells grown in medium 199 and 10% fetal calf serum without added test
fractions. In the graphic representation of the results, each point represents the mean
of readings from 6 wells.
Results
 Stepwise purification of the IgG fraction from plasma
resulted in a diminishing concentration of protein but a
gradually rising percentage of bioactivity among the
fractions.
2/27/03 EL 2+3: Dilution Curve
150.00%
Percent Bioactivity
140.00%
128.37%
130.00%
125.84%
123.88%
117.70%
120.00%
116.57%
113.76%
112.92%
110.00%
100.00%
100.00%
90.00%
80.00%
Control
1:100
1:400
1:1600
1:6400
1:25600
1:102400 1:409600
Dilution
The optimum bioactivity of the protein-A eluate was 128%. The
protein concentration of the eluate was determined to be .5466
mg/mL.
Bioactivity of Endothelial Cells with
2/27/03 Hydroxyapatite Test Fractions
170.00%
160.00%
151.71%
Percent Bioactivity
150.00%
147.44%
143.59%
140.00%
136.75%
132.91%
130.00%
120.00%
110.00%
100.00%
100.00%
90.00%
80.00%
Control
FT
50mM
100mM
250mM
400mM
Dilution
The 400 mM hydroxyapatite fraction eluted from a sample of
the eluate showed more bioactivity (149%) than the protein-A
eluate itself, while having a protein concentration of .2516
mg/mL. The 400mM fraction displayed more bioactivity with a
smaller amount of protein than the eluate.
Bioactivity of Endothelial Cells with
Fractions of the Immunoaffinity Column
bioactivity (endothelial cell assay)
protein conc
160.00%
0.1
0.08
0.07
140.00%
0.06
130.00%
0.05
0.04
120.00%
0.03
Protein
Concentration
(mg/mL)
Percent Bioactivity
0.09
150.00%
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
Fraction
In the next purification step, a 400mM fraction of the eluate was applied to an
immunoaffinity column. The fraction eluted at a pH of 3.3 displayed 153%
bioactivity with a protein concentration of .0129 mg/mL. This fraction
displayed more bioactivity with a substantially smaller amount of protein.
12/7/00 EL 2+3 Dilution Curve
140.00%
130.00%
Percent Bioactivity
127.12%
120.55%
120.00%
113.97% 112.88%
110.41%
110.00%
100.00%
107.95%
106.58%
100.00%
90.00%
80.00%
Control
1:100
1:400
1:1600
1:6400
1:25600
1:102400
1:409600
Dilution
To further characterize the antibody, a separate protein-A eluate was taken out to
different dilutions. The resulting dose-proliferation curves of bioactivity in
endothelial cells showed a sharp rise in activity at a 1:1600 dilution, and a
subsequent drop at higher dilutions. The activity of the antibody reached its peak at
a dilution of 1:1600.
Results
• When an older eluate (patient 89) was examined
with mass spec, the result was an extremely sharp
peak, which is an indicator of substantial purity.
• The peptide which induced this peak was of a
molecular weight of approximately 13 kDa.
• This peptide was subsequently sequenced and the
first 12 amino acids of it’s amino acid sequence
were determined.
Discussion
 As the IgG fraction was further purified the concentration of protein in
the purified samples was reduced as their bioactivity rose.
 This suggests that the methods use in conducting the purification were
successful in getting rid of proteins with inhibitory effects, thus
allowing the protein in the sample to be more active despite being at a
lower concentration.
 It is likely that this is because the inhibitory effects of other proteins
mask the biological activity of the bFGF-like antibody that is the
subject of the current research.
 Hence, samples that are less pure exhibit less bioactivity than purer
samples despite having higher protein concentrations.
Discussion
 The further purification of bFGF-like substances is an end toward
which the current study strives. Once the antibody of interest can be
isolated in a relatively pure state, it's amino acid sequence might be
determined just as some of the amino acid sequence of the older eluate
was.
 Determining the sequence of the peptide in the older eluate offers
strong evidence that the bioactivity of the fraction and others like it
that were used in laboratory experiments is indeed the result of an IgG.
 The derived amino acid sequence is probably the degraded part of the
variable region of one of the light chains from an IgG.
 Prior to this discovery, it was not unlikely that contamination of
samples by a small growth factor was the cause of bioactivity in
laboratory samples.
Acknowledgements
 I thank Dr. Mark B. Zimering, MD PhD, for his
intellect and good humor in the lab as well as
providing the opportunity to conduct this research.
 I thank Mrs. Jeanette Kim for her diligence in
making SRTP enjoyable.
 I thank Dr. Robert Donnelly, PhD for running our
laboratory samples over mass spectroscopy.
 I thank the VA for providing the laboratory
facility.