Fluorescent Cocaine Probes
A R T I C L E S
On the next day, the culture was spun down at 10 000g for 15 min.
The supernatant was transferred to a clean bottle, and 4% (w/v) PEG
8000 and 3% (w/v) NaCl were added. The phage were precipitated on
ice for 30 min. The precipitate was spun down at 8000g for 20 min.
The supernatant was discarded. The pellet was drained for 10 min and
then resuspended in 2 mL of PBS/1% BSA. The resuspended phage
were spun down at 14 000 rpm (Eppendorf microcentrifuge 5415C)
for 5 min. The supernatant contained the phage particles.
Star tubes (Nunc) were coated with GNC-BSA (100 µg/1 mL PBS/
tube) at 4 °C overnight. On the next day, the tubes were washed three
times with water and blocked with 5% dry milk in PBS for 1 h at 37
°C. The tubes were washed three times with dH2O, and 1 mL of the
phage suspension/tube was added. This was incubated at 37 °C for 1
h. After thorough washing with PBS/Tween 20 (0.05%), the bound
phage were eluted with 1 mL of elution buffer (0.1 M glycine, pH
2.3). The eluate was removed and neutralized with 12 µL of 2 M Tris
base (pH 8). Two milliliters of a fresh E. coli XL-1 blue culture (OD600
≈ 1) grown with tetracycline (10 µg/mL) was infected with the eluant.
After an incubation period of 30 min at room temperature, the culture
was inoculated into 100 mL of 2YT medium containing carbenicillin
(100 µg/mL) and tetracycline (10 µg/mL) and was incubated at 37 °C
for 4 h. Two milliliters of VCS M13 helper phage and 200 µL of IPTG
(1 mM) were added. After 30 min incubation at room temperature, the
culture was transferred to a shaker and shaken (300 rpm) for 90 min at
37 °C. After the addition of 140 µL of kanamycin (70 µg/mL), the
culture was incubated overnight at 30 °C. Phage particles were prepared
the next day as described (vide supra).
Protein Expression and Purification. Escherichia coli BL21 (DE3)
(Novagen) cells were transformed with the expression plasmid using
chemical transformation. On the next morning, 5 L of SB (3% peptone,
2% yeast extract, 1% MOPS) containing carbenicillin (100 µg/mL) was
inoculated. The cultures were incubated on a shaker (300 rpm) at 37
°C until an OD600 between 0.6 and 0.8 was reached. IPTG was added
up to a final concentration of 1 mM, and the temperature was adjusted
to 30 °C. The cultures were incubated overnight.
On the next morning, the cultures were chilled on ice for 30 min
and spun down at 8000g for 15 min. The supernatant was transferred
into clean flasks. In the next step, the supernatant was concentrated
using a Millipore concentrator. The volume of the supernatant was
reduced to approximately 250 mL. Affinity chromatography was applied
to purify the antibody fragment from the supernatant. The supernatant
was loaded onto a column containing M2-anti-FLAG resin (SIGMA).
After the loading, the column was washed thoroughly with PBS. The
scFv protein was eluted with elution buffer (0.1 M glycine, pH 2.3).
The eluate was neutralized with 1 M Tris (pH 9). The neutralized eluate
was concentrated with Centriprep YM10 concentrators (Amicon). The
final concentration of scFv protein was 1 mg/mL, which was monitored
as the absorption at 280 nm.
a high-affinity fluorescent probe for the selection of cocaine-
binding mAbs. From the standpoint of the selection of anti-
cocaine mAbs, but also fluorescent labeling of bioactive small
compounds in general, the effect incurred through a subtle
change in the linker was of great interest. Whereas fluorescent
probes GNC-F1 and GNC-I failed to show high affinity for
cocaine-binding mAbs, we did observe strong binding of several
antibodies to GNC-F2. Notably, we obtained apparent Kd
values that were close to the Kd values we obtained for these
mAbs when [3H]-cocaine was used in our equilibrium dialysis
binding assays. Using these assays, we were able to determine
Kd values for commercially available cocaine-binding mAbs,
as well as for a panel of scFvs. The affinity probe GNC-F2
could provide a powerful tool in selection and engineering
strategies for the production of high-affinity anti-cocaine mAbs
from a large panel of hybridoma cell cultures and phage-display
libraries. In contrast with [3H]-cocaine that presents problems
with respect to cost, handling, and waste management, the use
of the fluorescent probe GNC-F2 is more practical for the
characterization of large numbers of antibodies. In addition,
optical bioimaging to investigate structure and function of
proteins in cells and tissues continues to expand at a rapid pace.
For the fluorescence imaging of bioactive protein-ligand
interactions, the design of exogenous fluorescent labeling agents
and linkers attached to ligands could provide selectivity and
sensitivity of the imaging probes. GNC-F2 might serve as a
potential bioimaging probe to study the psychoactive and
addictive effects of cocaine in living tissue.
Experimental Section
Construction of a CDRH3 Walking Library. The gene encoding
the wild-type single chain variable fragment (scFv) antibody was used
as template in polymerase chain reactions (PCR). The gene fragments
containing the partly randomized CDRH3 were generated and amplified
using the primers MHVsfi (5′-ttgttattactcgcggcccagccggccatggca-3′),
CDRH3r (5′-ggtttcacagaaatatgtagccgtgtcc-3′),
CDRH3f (5′-gctacatatttctgtgaaaccnnknnknnknnknnknnkgactactgggg-
ccaaggcacc-3′), and MLJsfi (5′-gtcctcgtcgactggaattcggcccccgaggccac-
3′). The scFv gene with randomized CDRH3 was subsequently
assembled by overlap PCR using the gene fragments and finally
amplified using the primers MVHsfi and MLJsfi. The fusion PCR
products were digested with SfiI and ligated to SfiI-digested pCGMT
phagemid vector. The ligation mix was ethanol precipitated and
transformed into Escherichia coli XL-1 blue (Stratagene) by electro-
poration. After transformation, SOC (2% peptone, 0.5% yeast extract,
0.05% NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose) was im-
mediately added. The cells were allowed to recover at 37 °C for 1 h,
then plated onto Luria-Bertani (LB) agar plates containing carbenicillin
(100 µg/mL), tetracycline (10 µg/mL), and 20 mM glucose and
incubated at 30 °C overnight. The library was determined to consist of
3 × 107 members. On the following day, glycerol stocks of the library
were prepared and stored at -80 °C.
Panning of CDRH3 Walking Library. To amplify the CDRH3
walking library, 1 mL of glycerol stock was inoculated into a 1 L 2YT
(1.6% peptone, 0.5% yeast extract, 1% NaCl) containing carbenicillin
(100 µg/mL) and tetracycline (10 µg/mL). Upon reaching an optical
density at the wavelength of 600 nm (OD600) of ≈0.6, 1 mL of VCS-
M13 helper phage suspension (titer > 1013/mL) was added and
incubated at room temperature for 30 min. The culture was transferred
to a shaker (300 rpm) and shaken at 37 °C for 90 min. Finally,
kanamycin (70 µg/mL) and 200 µL of IPTG (1 mM) were added to
the culture. The temperature was adjusted to 30 °C and incubated
overnight.
Fluorescence Spectroscopy. A FluoroMax-2 spectrofluorometer
(Instruments S. A., Inc., Edison, NJ) equipped with a 150 W continuous
xenon lamp was used to measure fluorescence emission spectra and
quantum yields with both excitation and emission band-pass of 5 nm;
a PMT voltage of 50 V was used, and measurements were performed
at a scan rate of 1 nm/s. For the fluorescein-based probes, samples
were excited at λexc ) 485 nm and fluorescence was measured at λem
) 515 nm. For the N-methyl-isatoic amide probe, measurements were
performed at λexc ) 350 nm and λem ) 425 nm.
Binding Studies. Equilibrium dialysis was performed using either
[3H]-cocaine or a fluorescent cocaine probe as ligand and serial dilutions
of IgG or scFv antibodies as hosts (80 µL per well). Wells (12 per
sample) were then filled with another 80 µL of [3H]-cocaine in PBS (2
nM per well). A second plate was prepared with 2 × 12 wells containing
just PBS (160 µL/well). The two plates were tightly connected with
filled wells facing each other and separated with a dialysis membrane
(cutoff 6000-8000 Da). The plates were attached vertically to a shaker
and were shaken for 24 h at room temperature, after which they were
9
J. AM. CHEM. SOC. VOL. 127, NO. 8, 2005 2483
Meijler, Michael M.
