Citation
Thymosin β4 Secretion Due to Apoptotic Drugs in Macrophages and Fibroblasts

Material Information

Title:
Thymosin β4 Secretion Due to Apoptotic Drugs in Macrophages and Fibroblasts
Series Title:
Journal of Undergraduate Research
Creator:
Uryasev, Oleg
Bubb, Michael ( Mentor )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Language:
English

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Genre:
serial ( sobekcm )

Notes

Abstract:
Thymosin β4 (tß4), a 5 kDa protein, is a mediator of inflammation, with many functions in cellular motility, migration and damage repair. In spite of the fact that apoptotic cells exhibit pro-inflammatory signals, inflammation is absent during apoptosis. We believe that endogenous tß4 is released during apoptosis from cells such as macrophages and fibroblasts, in turn suppressing inflammation. Our primary aims for this project were to develop and refine tß4 detection assays/methods, mainly the ELISA. Competition fluorescence anisotropy was analyzed as a potential method for detecting tß4 in cellular supernatant. The RAW 264.7 macrophage-like cell supernatant contained an unknown factor interfering with exogenous tß4 added to cellular supernatant. We were unable to identify the interfering factor; however we were able to eliminate it through use of a protein concentrator, which likewise helped concentrate tß4 in individual samples. These findings lay the groundwork for further investigation using apoptotic drugs as a tß4 releasing agent in order to suppress inflammation.

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University of Florida
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University of Florida
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All applicable rights reserved by the source institution and holding location.

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Thymosin 34 Secretion Due to Apoptotic Drugs

in Macrophages and Fibroblasts


Oleg Uryasev


College of Liberal Arts and Sciences, University of Florida


Thymosin 34 (tM4), a 5 kDa protein, is a mediator of inflammation, with many functions in cellular motility, migration and damage
repair. In spite of the fact that apoptotic cells exhibit pro-inflammatory signals, inflammation is absent during apoptosis. We believe
that endogenous tM4 is released during apoptosis from cells such as macrophages and fibroblasts, in turn suppressing inflammation.
Our primary aims for this project were to develop and refine tM4 detection assays/methods, mainly the ELISA. Competition
fluorescence anisotropy was analyzed as a potential method for detecting tM4 in cellular supernatant. The RAW 264.7 macrophage-
like cell supernatant contained an unknown factor interfering with exogenous tM4 added to cellular supernatant. We were unable to
identify the interfering factor; however we were able to eliminate it through use of a protein concentrator, which likewise helped
concentrate tM4 in individual samples. These findings lay the groundwork for further investigation using apoptotic drugs as a tM4
releasing agent in order to suppress inflammation.


Thymosin 04 (tB4) is a 43 amino acid, 5 kDa
polypeptide with key roles in cell motility and
organogenesis [1,2]. In addition, tB4 has anti-inflammatory
properties as seen in recent studies, where corneas treated
with exogenous tB4 exhibited higher recovery rates and
decreased inflammation than those with a control of PBS
[3]. During another study, exogenous tB4 promoted
survival and repair of postnatal cardiomyocytes after
coronary ligation [4]. In these experiments, the effect of
necrotic cells, which ordinarily cause inflammation to the
surrounding living tissue, was diminished. Paradoxically,
in spite of the release of pro-inflammatory mediators
during apoptosis, apoptotic cells lack the inflammatory
properties observed during cellular necrosis.
Based on these observations, we hypothesized that
endogenous tB4 released from cells during apoptosis
suppresses the inflammatory response. A long-term
research objective for this project is to determine drugs that
trigger apoptosis, and subsequently cause cells to release
tB4. These results could form the basis for development of
new anti-inflammatory drugs. The FDA approved drug
Paclitaxel, used against cancer, is one such an example of
an apoptotic agent [5].
tB4 is present in high concentrations in macrophages
and fibroblasts [6,7]. Macrophage-like RAW 264.7 and
fibroblast NIH-3T3 cell lines were used to study tB4
release from apoptotic cells. Our hypothesis was that these
cells treated with apoptotic drugs, such as Taxol or
Etoposide, undergo apoptosis and release tB4 in the
extracellular media. Assays for detection of apoptosis,
including measurements of Caspase-3 activation and


Annexin V exposure, were considered as a method to
determine the percent of apoptotic cells.
Enzyme-Linked Immuno-Sorbant Assays (ELISA)
were used to detect the concentration of tB4 in cell
supernatants. Based on our previous hypotheses, a positive
correlation between the extent of apoptosis and the
concentration of tB4 in the media was anticipated. tB4
exhibits extracellular activities at very low concentrations
which indicates that tB4 released into cellular media could
be correspondingly small [8,9]. Therefore, quantitative and
qualitative techniques for detection of tB4 should be very
sensitive. The possibility exists that ELISA assays are not
sensitive enough to detect nanomolar concentrations at
which tB4 is observed to have activity.

METHODS
Cell cultures
RAW 264.7 cells were a generous gift from Dr.
Shannon Holliday's Lab (University of Florida,
Gainesville, Fl). The cells were grown in Dulbecco's
modified Eagle's medium with 4 mM L-glutamine adjusted
to contain 1.5 g/L sodium bicarbonate, 20 mg/L
gentamycin and 4.5 g/L glucose, 90%; fetal bovine serum,
10%. Cells were cultured in 25 cm2 plates and transferred
once 80% confluency was reached.
Confluent 25 cm2 flasks were scraped and frozen in a
Styrofoam slow freeze contraption in DMEM/10% FBS
supplemented with 5% DMSO at -800C. Frozen cell vials
were then transferred and stored in liquid nitrogen. Each
freezer stock yielded enough for two plates when thawed.


University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 I Spring 2009





OLEG URYASEV


Cell treatment with Paclitaxel
RAW 264.7 cells were treated with Paclitaxel (Taxol)
0-1000 nM diluted in 'no FBS' DMEM for a period of 10
hours at 370C in 25cm2 flasks. Controls were treated with
'no FBS' DMEM.

Standard ELISA
Immulon 4HBX ELISA plate was coated with 300 gl
of recombinant tB4 in 'No FBS' DMEM media containing
Dulbecco's modified Eagle's medium (with 4 mM L-
glutamine adjusted to contain 1.5 g/L sodium bicarbonate,
20 mg/L gentamycin and 4.5 g/L glucose) and incubated at
220C for 60 minutes. The plate was washed three times
with PBS (0.43 mM Na2PO4, 0.27 mM KC1 13.7 mM
NaC1), 0.02% Tween20 and blotted dry with a paper towel.
Blocking buffer (5% BSA, 100 mM L-Lysine, pH 7.0) was
added to each well and incubated at 220C for 60 minutes.
The plate was washed three times with PBS, 0.02%
Tween20 and blotted dry with a paper towel. Primary
antibody (monoclonal mouse anti-tB4 produced in the
University of Florida core facility) in PBS was added and
incubated overnight at 40C. The plate was washed three
times with PBS, 0.02% Tween 20 and blotted dry with a
paper towel. Secondary antibody (alkaline phosphatase
mouse IgG, Jackson Labs, 115-055-003) in 0.01M Tris,
0.25 M NaCl was added and incubated at room temperature
for 60 minutes. The plate was washed five times with 0.01
M Tris, 0.25 M NaCl and blotted dry with paper towel.
Developing solution (5 ml DI H20, 2.5 .il 1M MgCl2, 5 .il
diethanolamine, 5 mg tablet of pNPP (Sigma S0942)) was
added to each well. The plate was incubated at 220C until
color was visible. OD was read on a SpectraMax 5
spectrophotometer (Toronto, Canada) at 405 nm.

Fluorescence anisotropy
Data were collected using serial dilutions of samples
in 0.3 ml in glass cuvettes with a Photon Technology
International (South Brunswick, NJ) spectrofluorimeter.
The Rhodamine labeled tB4 (Rh-tB4) was excited with a
vertically polarized light at a wavelength of 546 nm, with
horizontal (Ih) and vertical (Iv) components of the emitted
light measured at 575 nm for -30 s for each sample.
Fluorescence anisotropy (r) was calculated using r = (I -
GIh ) /(Iv + 2GIh ). The G factor for each experiment was
determined by exciting the labeled peptide in solution with
a horizontally polarized light [10] and averaged over -10
measurements. For the direct binding assay, anisotropy was
measured as a function of mouse anti-tB4 concentration
with a Rh-tB4 concentration of 10 nM. The observed
anisotropy was a linear function of the concentration of
Rh-tB4 bound to the mouse anti-tB4 antibody because total
intensity of recombinant Rh-tB4 fluorescence (I + 2GIh)
did not significantly change upon binding to monoclonal
mouse anti-tB4 [11].


Rhodamine-labeled t134 binding to plate
60 gl of Rhodamine labeled tB4 was incubated in 'no
FBS' DMEM or sample for 60 minutes at 220C. Excitation
at a wavelength of 530 nm and emission at 570 nm was
measured on a spectrophotometer (SpectroMax 5) prior to
incubation, after incubation, and after three washes of PBS.

Tubulin ELISA
Method described above under standard ELISA.
Tubulin (Cytoskeleton, ML113) was used in place of the
recombinant tB4. Mouse Anti-Tubulin antibody (Sigma,
T5168) was used in place of primary monoclonal mouse
anti-tB4 antibody.

Protein concentration
Cell-free supernatant and 'no FBS' DMEM were each
adjusted to contain recombinant t34 (33.3 nM, 1 ml total
volume). Each sample was loaded onto a Centricon-3
concentrator (Amicon, 42403) and spun for 45 minutes at
7500x g on a table-top centrifuge. Samples were washed by
adding 1 ml of PBS to the concentrate. Samples were
centrifuged again for 45 minutes at 7500x g. Initial flow-
through and final concentrate were removed and analyzed
using an ELISA assay.

RESULTS
A potential alternative method for tf34 detection.
A fluorescence anisotropy assay was used to
determine affinity of monoclonal mouse anti-tB4 affinity to
Rhodamine labeled tB4 (Rh-tB4). Increasing concentrations
of monoclonal anti-mouse tB4 were added to Rh-tB4 (10
nM) until a maximal binding was achieved (Fig. 1). Direct
binding data showed that tB4 has a low affinity for
monoclonal mouse anti-tB4, with a Kd of 380 nM (Fig. 1).
Kd is the equilibrium dissociation constant which has an
inverse correlation to affinity. Using Osmolyte
trimethylamine-N-oxide (TMAO), a molecular crowding
agent, is a potential strategy to increase affinity and
decrease Kd. The affinity of Rh-tB4 to mouse anti-tB4,
while increased (Kd = 120 nM), was still low (Fig. 1).

Contents ofRAW 264.7 cell-free supernatant interfere with
the identification/quantification of endogenous t/4 using
an ELISA assay
In developing a standard ELISA for tB4 using
recombinant protein, a sensitivity of 5 pmol was
determined (Fig. 2). Macrophage-like RAW 264.7 cells
were treated with Paclitaxel and cellular supernatants were
assayed for tB4 using an ELISA assay (Fig. 2). No tB4 was
detected in the cellular supernatants with standard ELISA.
The effect of cell-free supernatant on the sensitivity of
ELISA detection was tested using modified tp4 ELISA
protocol (Fig. 3) as described below. Supernatant was
collected from RAW 264.7 cells exposed


University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 I Spring 2009





THYMOSIN B4 SECRETION


100

80

60

40 - Thymosin p4
0 PBS K = 380 nM
20 - 1.5MTMAO K = 120 nM)

0


0 1 2 3
Antibody (pM)


4 5


Fig. 1. Direct binding of monoclonal mouse anti-tR4 to Rhodamine labeled
(Rh-tR4) using fluorescence anisotropy. Increasing concentrations of
monoclonal anti-mouse tR4 were added to Rh-tR4 (10 nM) diluted in PBS
buffer. Monoclonal mouse anti-tR4 has a low affinity for tR4, with a Kd of
380 nM. The same assay was performed again in presence of at 1.5 M
osmolyte trimethylamine-N-oxide (TMAO), a molecular crowding agent.
TMAO was a potential strategy to increase affinity and decrease Kd,
however the affinity of Rh-tR4 to mouse anti-tR4, while increased (Kd =
120 nM), was still low. These data indicate that competition fluorescence
anisotropy would not provide enough sensitivity with this monoclonal
mouse anti-tR4 antibody.

to 'no FBS' DMEM for 1 hour. The standard ELISA
protocol described in Methods section was modified by
including an extra 1 hour 'preincubation' prior to the
normal 'incubation' with recombinant tB4 or treated cell
sample. The following were performed in duplicate with
three PBS washes between each preincubation and
incubation. (1) Preincubation: cell-free supernatant.
Incubation: 66.6 nM tB4 in 'no FBS' DMEM (2)
Preincubation: 'no FBS' DMEM. Incubation: cell-free
supematants adjusted to 66.6 nM recombinant tB4 (3)
Preincubation: 66.6 nM tB4 in 'no FBS' DMEM.
Incubation: cell-free supernatant (4) Preincubation: 'no
FBS' DMEM. Incubation: 0 or 66.6 nM recombinant tB4 in
no FBS' DMEM. Whenever the supernatant was
incubated with the tB4, or prior to the tB4, an inhibition of
OD signal at 405 nm occurred. The absorption dropped
from 66.6 nM levels to 0 nM levels. In the samples where
supernatant was added after tB4 and allowed to attach to
the plate, the OD signal was uninhibited. The observed
inhibition of OD signal was confirmed by repeating the
same experiment in triplicate (data not shown).


Contents of RAW 264.7 cell-free supernatant prevent the
binding ofRh-tJ34 to ELISA plate
Rhodamine labeled tB4 was used to confirm
whether tB4 bound to the ELISA plate in the presence of
cell-free supernatant (Fig. 4). Fluorescence of Rh-tB4 was a
linear function of its concentration, as expected, when 60
gl volume samples of various tB4 concentrations


2U
18
16
14
S12


a 08
08
06
04 *
02
00
0 20 40 60 80 100 120 140
tB4 (nM)


20
18
16
14
1 2
1 0
08
06
04 m -
02
00
0 200 400 600 800 1000
Taxol (nM)
Fig. 2. ELISA of recombinant tR4 in 'no FBS' DMEM and supernatant
derived from Paclitaxel treated RAW 264.7 cells. Standard ELISA protocol
described in Methods was followed for this assay. Paclitaxel treated (0-
1000 nM) RAW 264.7 cells showed no visible tR4 in their supernatant
which could be due to an inhibitor in the supernatant or a concentration of
tR4 too low to detect with the ELISA.

were added to an ELISA plate and read on a
spectrophotometer (Fig. 4A). A maximum of 2 pmol Rh-
tB4 remained bound to the plate after three washes with
PBS regardless of the initial concentration loaded. Rh-tB4
was incubated on an ELISA plate in the presence or
absence of cell-free supernatant (Fig. 4B). Cell-free
supernatant and 'no FBS' DMEM were each adjusted to
contain 1.6 ipM Rh-tB4 and incubated on an ELISA plate
for one hour. Emission from the samples was identical
before and after the incubation. The ELISA plate was
subsequently washed with PBS as per standard ELISA
protocol and read for emission on the spectrophotometer.
The cell-free supematant/Rh-tp4 samples had lower
emission as compared to the 'no FBS' DMEM/Rh-tp4
samples.

Interfering component of RAW 264.7 cell-free supernatant
not specific for t34
In order to remove the inhibitor from the supernatant,
an important step was to determine whether the inhibition
was specific to tB4, or whether another ELISA would have


University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 I Spring 2009





OLEG URYASEV


0 * cell-free supernatant
incubated prior to tB4
AtB4 incubated prior to
cell-free supernatant
05 cell-free supernatant
incubated with tB4
*tB4 control cure
04
E

0 03
0
02

01


50 100 150 200 250
tP4 (nM)


300 350 400


Fig. 3. Modified ELISA assay designed to reveal inhibition effects of cell-
free supernatant. Standard ELISA protocol was modified to include a 1
hour 'preincubation' (P) before incubation (I) step. The samples were
incubated as follows- (1) P: cell-free supernatant. /: 66.6 nM tR4 in 'no
FBS' DMEM, (2) P: 66.6 nM tR4 in 'no FBS' DMEM. /: cell-free
supernatant, (3) P: 'no FBS' DMEM. /: cell-free supernatants adjusted to
66.6 nM recombinant tR4 (4) P: 'no FBS' DMEM. /: 0 or 66.6 nM
recombinant tR4 in 'no FBS' DMEM. After the plate was saturated with
recombinant tR4, cell-free supernatant could not inhibit ELISA absorbance
and therefore, the inhibitor does not displace tR4 bound to the plate or
form a complex unrecognizable by primary mouse anti-tR4.



inhibition as well. Using the developed standard ELISA
protocol, an ELISA to an unrelated protein, Tubulin, was
developed (Fig. 5A). The ELISA plate was
preincubated with 'no FBS' DMEM or cell-free
supernatant. Tubulin was incubated in 1.6 pM
concentration. When the cell-free supernatant was
preincubated, the signal from Tubulin was inhibited by
50% as compared to the 'no FBS' DMEM preincubation.


Large cellular debris and lipids from RAW 264.7 cell-free
supernatant are not responsible for tf34 ELISA inhibition
In order to remove the possibility that large cellular
debris or lipids were responsible for ELISA inhibition,
RAW 267.4 cell-free supernatants were exposed to 800C
for 10 minutes, followed by centrifugation at 17,746x g for
10 minutes on a table-top centrifuge (Fig. 6). The treated
supematants were preincubated on an ELISA plate,
followed by 33.3 nM recombinant tp4 in 'no FBS' DMEM.
Control wells were preincubated with 'no FBS' DMEM
followed by 33.3 nM recombinant tp4 in 'no FBS' DMEM.
High temperature and centrifugation failed to remove the
inhibitory component from the cell-free supernatant.


Inhibitor from RAW 264.7 cell-free supernatant separated
using Centricon-3 concentrator
To eliminate small molecules in the cell-free
supernatant, cell-free supernatant with recombinant tp4
was concentrated and washed using a protein concentrator
with upper cutoff of 3kDa (Fig. 7). The following


2000
1800
1600
1400
1200
1000
800
600
400
200 -
O ii---
0 1000 2000 3000 4000
Rh-tB4 (nM)


18
16
14
12
:10
8
6
4
2

0 1000 2000 3000 4000
Rh-tB4 (nM)


O1000
l)
-

o 800
Ln
S600

B 400

o 200
5
o 0
LL


'Before wash'


S'1.6mkM Rho-lab
tB4 in no phenol
red DMEM'
S'1.6mkM Rho-lab
tB4 in
supernatant'







E'1.6mkM Rho-
lab tB4 in no
phenol red
DMEM'
E'1.6mkM Rho-
labtB4 in
supernatant'


'After wash'


Fig. 4. Rhodamine labeled tR4 (Rh-tR4) binding to ELISA plate in the
presence of cell-free supernatant. The Rh-tR4 fluorescence was a linear
function of Rh-tR4 concentration when added to an ELISA plate and read
on a spectrophotometer at Excitation: 530nm, Emission: 570nm (A). A
maximum of 2 pmol of Rh-tR4 remained bound to the plate after three
washes with PBS regardless of the initial concentration loaded (B). Cell-
free supernatant and 'no FBS' DMEM were each adjusted to contain 1.6
pM Rh-tR4 and incubated on an ELISA plate (C). After wash, the cell-free
supernatant/Rh-tp4 samples had lower emission as compared to the 'no
FBS' DMEM/Rh-tp4 samples, indicating that Rh-tp4 was unable to bind to
the ELISA plate in the presence of cell-free supernatant (D).


University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 I Spring 2009





THYMOSIN B4 SECRETION


1 6 Control curve tubulin
1 4 * Pre-incub with 36hr notaxd
media
12
1

08
0 06
04
02
0
0 01 02 03


04 05 06 07 08
Tubulin (ng)


14

12

1
E
S08

S06
O
0
04

02

0


09 1


0 10 20 30 40
tB4 (nM)


50 60 70


Fig. 5. Component in RAW 264.7 cell-free supernatant inhibits the
standard ELISA assay for recombinant tubulin. The standard ELISA
protocol was adapted for tubulin by using recombinant tubulin and
monoclonal mouse anti-a-tubulin. Preincubation with cell-free supernatant
as compared to 'no FBS' DMEM caused a 50% inhibition of 1.6 pM tubulin
signal, and demonstrated that the inhibitor was non-specific to tR4.


Kcell-free supernatant
1 r 2 - rir c b


Fig. 7. ELISA of concentrates and flow-throughs from RAW 264.7 cell free
supernatant and 'no FBS' DMEM adjusted to contain recombinant t34
(33.3 nM, 1 ml total volume). Each sample (1 ml of volume) was loaded
onto a Centricon-3 concentrator (Amicon, 42403) and concentrated at
7500x g on a table-top centrifuge with 1 ml of PBS as a wash step.
Control recombinant tR4 (0, 33.3 nM and 66.7 nM) was diluted in 'no FBS'
DMEM. All shown data is in duplicate. The Centricon-3 concentrator was
efficient in removing the inhibitor from the cell-free supernatant however
was not highly efficient for tR4 concentration.


Using the concentrate and flow-through samples, an
-cell-free supernatant with ELISA was performed. Control recombinant tB4 (0, 33.3
heat and centrifuge
incubated prior to tB4 nM and 66.7 nM) was diluted in 'no FBS' DMEM and
+Control tB4 curve showed a linear correlation as expected. Recombinant tB4

with 'no FBS' DMEM and in cell-free supernatant in the
Centricon-3 showed increase of tB4 to 66.7 nM in the
concentrate as compared to the control (33.3 nM).
Recombinant tB4 with 'no FBS' DMEM flow-through
showed an equal amount of tB4 as compared to the control.
0 5 10 15 20 25 30 35 Uncentrifuged supernatant had a decreased signal of -80%
tp4 (nM) (down to 5 nM) as compared to the control (33.3 nM).
Recombinant tB4 with supernatant flow-through showed
LISA of RAW 267.4 cell-free supernatants exposed to 80'C for 100% (0 nM) inhibition of protein signal as compared to
es, followed by centrifugation at 17,746 x g as compared to the control.


control recombinant tS4 in 'no FBS' DMEM. Supernatant was
preincubated followed by 33.3 nM recombinant tP4 in 'no FBS' DMEM.
Control tS4 wells were preincubated with 'no FBS' DMEM. The
centrifugation and heat samples still inhibited the signal from tS4 in the
ELISA, therefore indicating large cellular debris and lipids were not
responsible for ELISA inhibition

experiment was performed in duplicate: RAW 264.7 cell
free supernatant was adjusted to contain recombinant tp4
(33.3 nM, 1 ml total volume). A control of 'no FBS'
DMEM was adjusted to contain recombinant tp4 (33.3 nM,
1 ml total volume). Each sample (1 ml of volume) was
loaded onto a Centricon-3 concentrator (Amicon, 42403)
and spun for 45 minutes at 7500x g on a table-top
centrifuge. Initial flow-through was removed and saved for
an ELISA assay. 1 ml of PBS was added to the concentrate
as a wash step. Samples were spun again for 45 minutes at
7500x g (until 100 pil were left of concentrate).


DISCUSSION


Working under the hypothesis that endogenous tB4, a
known anti-inflammatory agent, is released during
apoptosis, therefore preventing inflammation, one of the
primary goals of our project is to develop methods for
detecting tB4 in cell free supematants of apoptotic cells.
The ELISA assay was used as a potential methods of
detecting endogenous tB4 and increasing sensitivity to tB4
was the initial step. The lowest detectable recombinant tB4
concentration was 5 pmol as seen in the ELISA. tB4 has
anti-inflammatory effects at low nM and even pM
concentrations and therefore, our sensitivity of 20 nM for
ELISA is possibly not high enough to detect
physiologically relevant release of tB4 [8,9].


University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 I Spring 2009


* Control tB4

* Conc Supernatant with
tB4
Flow-Through
Supernatant with tB4
Supernatant with tB4

xConc 'no FBS' DMEM
wth tB4
* Flow-Through 'no FBS'
DMEM wth tB4


1

08

06

04


Fig. 6. EL
10 minute





OLEG URYASEV


Competition fluorescence anisotropy measures native tB4
concentration in media or buffer by using fluorescent
Rhodamine-labeled tB4 (Rh-tB4) to compete for the same
binding site on an antibody. With increasing concentrations
of unlabeled tB4, the signal from Rh-tB4/antibody complex
diminishes until a saturation point with native tB4 is
reached, and all of the Rh-tB4 is displaced. In order to
effectively measure low tB4 concentrations in a sample, an
antibody with high affinity to an antigen is necessary.
Direct binding fluorescence anisotropy assay was used to
determine the affinity of monoclonal mouse anti-tp4
antibody for tp4, which turned out to be low (Fig. 1). With
the addition of a molecular crowding agent, TMAO, the
affinity increased 3 times, although this is not enough. The
low affinity of monoclonal mouse anti-tp4 suggests that
competition fluorescence anisotropy using this particular
antibody is not an effective method for identifying and/or
quantifying tp4 in cell-free supernatants.
Supernatant from RAW 264.7 cells treated with
Paclitaxel, a known apoptotic agent, was assayed in the tB4
ELISA mentioned above. Contrary to our expectations, no
appreciable tp4 signals in the supematants could be
detected as compared to the control, with recombinant tp4
in 'no FBS' DMEM (Fig. 2B). A possible explanation is
that tB4 concentrations in cell-free supernatants with and
without taxol treatment were lower than ELISA sensitivity.
However, when tB4 was added to the cell-free supematants
and analyzed in an ELISA for control purposes, an
inhibitory factor was found (Fig. 3).
We stipulated the following explanations as to why
tB4 was not detected in supernatants by the ELISA assay.
A component of the cell supernatant has some effect on the
ELISA and could be acting to inhibit the assay by: (1)
displacing tB4 bound to the ELISA plate, (2) forming a
complex with tB4 which cannot bind to the plate surface,
(3) binding to tB4 and forming a complex which cannot
interact with the primary mouse anti-tB4 antibody, (4)
binding to the plate, blocking all available tp4 binding
sites.
The ELISA protocol was modified to include a 1 hour
preincubation step prior to the initial incubation. Incubating
supernatant prior to, with, and after tB4 revealed the affects
of supernatant on the ELISA. After the plate was saturated
with recombinant tB4, cell-free supernatant could not
inhibit ELISA absorbance. Therefore, the inhibitor neither
displaces tB4 bound to the plate nor forms with tB4 a
complex unrecognizable by primary mouse anti-tB4. When
supernatant was added to the plate to which tb4 was
already bound, it could neither displace tb4 nor form a
complex unrecognizable by primary mouse anti-tB4. In the
experiment when cell supernatant was incubated in the
plate prior to addition of tb4, it was washed out before tb4
addition and therefore never contacted thb4 directly in
solution. Yet it could still inhibit ELISA. Therefore the


hypothesis for forming a complex with tB4 which cannot
bind to the plate surface also seems unlikely.
In order to determine whether the tB4 ELISA was
inhibited at the binding step, Rh- tB4 was plated in the
presence of cell-free supernatant. The cell-free
supernatant/Rh-tp4 samples had lower emission as
compared to the 'no FBS' DMEM/Rh-tp4 samples,
indicating that Rh-tp4 was unable to bind to the ELISA
plate in the presence of cell-free supernatant (Fig. 4).
Determination of whether the ELISA inhibition was
specific for tB4 was accomplished through an ELISA of a
non-related protein using supernatant to preincubate the
wells. The developed Tubulin ELISA demonstrated an
inhibition of signal from a component of the cell-free
supernatant, and therefore, that the interaction with tB4 was
non-specific (Fig. 5). Large cellular debris and lipids were
still present in supernatant and were possibly responsible
for ELISA inhibition. Heating and centrifugation of
supernatant did not remove the inhibitory affects from the
supernatant, therefore indicating large cellular debris and
lipids were not the causative agent in decreased signal (Fig.
6).
The Centricon-3 concentrator was efficient in
removing the inhibitor from the cell-free supernatant;
however, it was not highly efficient for tB4 concentration
(Fig. 7). The concentration of tB4 increased only twice,
whereas the volume was decreased ten times, indicating a
large amount (80%) of tB4 (5kDa) passed through the 3kDa
pores. Since the interfering was washed away through
3kDa pores of the cellulose acetate membrane, most likely
the inhibitor was a small molecule which interacted with
the ELISA plate to prevent tB4 binding to the plate.



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University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 I Spring 2009





THYMOSIN B4 SECRETION

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University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 | Spring 2009