Generation of novel single-chain antibodies by phage-display technology to direct imaging agents highly selective to pancreatic {beta}- or {alpha}-cells in vivo.

Generation of Novel Single-Chain Antibodies by
Phage-Display Technology to Direct Imaging Agents
Highly Selective to Pancreatic �- or �-Cells In Vivo
Sandra Ueberberg,1 Juris J. Meier,2 Carmen Waengler,3,4 Wolfgang Schechinger,1
Johannes W. Dietrich,1 Andrea Tannapfel,5 Inge Schmitz,5 Ralf Schirrmacher,4 Manfred Ko¨ller,6
Harald H. Klein,1 and Stephan Schneider1
OBJECTIVE—Noninvasive determination of pancreatic �-cell
mass in vivo has been hampered by the lack of suitable �-cell–
specific imaging agents. This report outlines an approach for the
development of novel ligands homing selectively to islet cells in
vivo.
RESEARCH DESIGN AND METHODS—To generate agents
specifically binding to pancreatic islets, a phage library was
screened for single-chain antibodies (SCAs) on rat islets using
two different approaches. 1) The library was injected into rats in
vivo, and islets were isolated after a circulation time of 5 min. 2)
Pancreatic islets were directly isolated, and the library was
panned in the islets in vitro. Subsequently, the identified SCAs
were extensively characterized in vitro and in vivo.
RESULTS—We report the generation of SCAs that bind highly
selective to either �- or �-cells. These SCAs are internalized by
target cells, disappear rapidly from the vasculature, and exert no
toxicity in vivo. Specific binding to �- or �-cells was detected in
cell lines in vitro, in rats in vivo, and in human tissue in situ.
Electron microscopy demonstrated binding of SCAs to the endo-
plasmatic reticulum and the secretory granules. Finally, in a
biodistribution study the labeling intensity derived from [125I]-
labeled SCAs after intravenous administration in rats strongly
predicted the �-cell mass and was inversely related to the
glucose excursions during an intraperitoneal glucose tolerance
test.
CONCLUSIONS—Our data provide strong evidence that the
presented SCAs are highly specific for pancreatic �-cells and
enable imaging and quantification in vivo. Diabetes 58:2324–
2334, 2009
The ability to noninvasively measure pancreatic�-cell mass in vivo may provide an early diag-nostic tool for the diagnosis of type 1 diabetesand potentially promote the development and
evaluation of novel therapeutic strategies. However, de-
spite enormous efforts by various groups, none of the
approaches tested have permitted a reliable and noninva-
sive assessment of �-cell mass in humans.
The small size of islets (50–300 �m in diameter), their
low abundance (1–2% of pancreatic mass), and their
scattered distribution throughout the pancreas create
technical challenges for noninvasive imaging of �-cell
mass. Because pancreatic islets possess no intrinsic con-
trast from the surrounding exocrine tissue, imaging tech-
niques have focused on detecting the pancreatic islet with
exogenous agents that are preferentially bound or concen-
trated in islets, and an extensive library of radiolabeled
agents has been applied to islet imaging. However, radio-
labeled compounds targeting the sulfonylurea receptor
(1–5), the presynaptic vesicular acetylcholine transporter
(6), and dopamine uptake in synaptic vesicles (7) have not
proven to be effective for islet imaging, most likely be-
cause of their failure to achieve the required endocrine-to-
exocrine binding ratio (�100:1), as suggested by Sweet et
al. (8,9). Another proposed strategy to image islets within
the pancreas relates to radiolabeled peptides derived from
glucagon-like peptide 1 (GLP-1) or its analogues, taking
advantage of the observation that the GLP-1 receptor is
expressed at a high density in the islets, but not in
exocrine cells (10). This approach has proven useful for
imaging insulinomas (11), but has not yet been demon-
strated to allow for the determination of islet cell mass in
healthy humans or patients with diabetes. Harris and
colleagues used [11C]DTBZ, a ligand to vesicular mono-
amine transporter 2 (VMAT2), to image pancreatic islets in
rodents and humans (12–14) by the means of positron
emission tomography (PET). Although initial results with
DTBZ have been rather encouraging (12,13), recent find-
ings suggest that nonspecific, non–�-cell binding of
[11C]DTBZ in the pancreas may limit its utility as a �-cell
imaging agent in humans (14).
An obvious reason for these difficulties in generating a
valuable imaging agent is the paucity of knowledge about
potential targets that are exclusively expressed on the
�-cell surface. One way to overcome these limitations is to
use phage-display technology, a powerful approach for
isolating target-specific peptides and antibodies in animals
and humans (15,16). Such single-chain antibodies (SCAs)
From the 1Department of Internal Medicine I, Division of Endocrinology and
Metabolism, Berufsgenossenschaftliches University Hospital Bergmann-
sheil, Ruhr-University Bochum, Bochum, Germany; the 2Department of
Internal Medicine I, St. Josef-Hospital, Ruhr-University Bochum, Bochum,
Germany; the 3Department of Nuclear Medicine, Hospital of the Ludwig-
Maximilians-University, Munich, Germany; the 4Department of Neurology &
Neurosurgery, Lady Davis Institute for Medical Research, McGill University,
Montreal, Canada; the 5Institute for Pathology, Ruhr-University Bochum,
Bochum, Germany; and 6Chirurgische Forschung, Berufsgenossenschaftli-
ches Universita¨tsklinikum Bergmannsheil, Ruhr-Universita¨t Bochum, Bo-
chum, Germany.
Corresponding author: Stephan Schneider, stephan.schneider@ruhr-uni-
bochum.de.
Received 4 May 2009 and accepted 3 July 2009.
Published ahead of print at http://diabetes.diabetesjournals.org on 10 July
2009. DOI: 10.2337/db09-0658.
© 2009 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
ORIGINAL ARTICLE
2324 DIABETES, VOL. 58, OCTOBER 2009 diabetes.diabetesjournals.org

that trigger receptor endocytosis directly have previously
been isolated by recovering infectious phages from within
cells after receptor-mediated endocytosis (17). For exam-
ple, internalizing SCAs targeting erbB2 and epidermal
growth factor receptor have been identified and used to
specifically deliver drugs into breast cancer cells (18,19).
Therefore, in the present study, we applied repeated phage
panning and rescued the bound phages from within the
islet cells to isolate SCAs specifically internalizing in islet
�-cells. These SCAs were then further characterized and
evaluated with respect to their suitability for islet imaging
purposes.
RESEARCH DESIGN AND METHODS
Animal model and human tissue samples. Female CD-rats (6 weeks of age;
200–250 g) received a single intraperitoneal injection of streptozotocin (STZ)
14 days before experiment. To induce different amounts of �-cell mass
reduction, rats were treated with either 30 mg/kg (low dose; n � 5) or 60
mg/kg (high dose; n � 5) STZ. All animal studies were approved by the
Landesamt fu¨r Naturschutz (Recklinghausen, Germany, no. 50.10.32.08.037).
The use of nondiabetic human pancreatic tissue samples was approved by the
ethics committee of the Ruhr-University Bochum (no. 2528, amendment 3).
Phage library. The recombinant phage library Tomlinson I is constructed in
the pIT2 vector (derived from pHEN1), consists of about 1.4 � 108 different
human single-chain variable fragments, and was provided by MRC Geneser-
vice (Cambridge, U.K.). The library is based on a single human framework for
VH (V3–23/DP-47 and JH4b) and V (O12/O2/DPK9 and J 1) with diversified
(DVT) side chains incorporated in complementarity-determining region (CDR)
2 and CDR 3.
Phage library screening. The library was screened for islet-specific SCAs
using two approaches. 1) For in vivo screening, a nondiabetic CD-rat was
injected intravenously through the jugular vein with 1012 phage transducing
units and allowed to circulate for 5 min. The pancreas was removed, and to
recover islet bound phages, the islets were isolated according to a standard
protocol except the use of FCS (20). 2) For in vitro screening, directly isolated
rat islets were incubated with 1012 phage transducing units for 1 h at 37°C in
vitro, as described previously (21). Subsequently, islets from both approaches
were treated equally to rescue phages from within the islet cells. Briefly, the
islets were washed twice by suspension in 1 ml PBS, followed by lysing the
cells by the addition of 1-ml hypotonic solution (30 mmol/l Tris-HCl, pH 8.0)
and a single freeze–thaw cycle. Subsequently, the suspension was incubated
overnight with TG1 bacteria at 37°C, with transducing units determined to
monitor the progress of each round of library panning and the phages
amplified according to standard protocols for use in the next round (22).
Purification of SCAs. HB2151 bacteria (OD600 � 0.4) were infected with 10
�l of isolated phage clones of interest, and colonies were further grown in 2 �
TY (100 �g/ml ampicillin and 1% glucose). Isopropyl �-D-thiogalactoside
(IPTG; AppliChem, Darmstadt, Germany) was added to induce SCA expres-
sion. The supernatant containing the SCAs was purified by metal affinity
chromatography (Nunc ProPur, Nunc, Langenselbold, Germany). The purity
of the sample was checked by SDS gel electrophoresis and Western blotting.
Immunohistochemistry. Staining of formalin-fixed, paraffin-embedded rat
and human tissue sections (5 �m) was performed as follows: Sections were
deparaffinized using xylol twice for 10 min and followed by EtOH thrice for 5
min and Aqua dest. for another 5 min. Afterward, the sections were perme-
abilized by heating in the microwave in antigen unmasking solution pH 6 and
down cooling for 45 min. Blocking was done for 1 h at 24°C with PBS
containing 2% BSA. Primary and secondary antibodies were diluted in PBS
with 2% BSA. Primary antibodies were incubated at 4°C overnight, except for
insulin and glucagon with an incubation period of 1 h at 37°C. Secondary
antibodies were incubated for 30 min at 24°C, and the same holds true for the
Cy2- and Cy3-conjugated streptavidin reagents. The following primary anti-
bodies and dilutions were used: SCA B1 and SCA A1, 1:200; monoclonal mouse
anti-c Myc antibody, 1:200 (Cell Signaling, 2276); polyclonal guinea pig anti-
swine insulin antibody, 1:400 (Dako, A0564); and monoclonal mouse
antiglucagon antibody, 1:200 (Affinity BioReagents, MA1-20210). Secondary
antibodies were monoclonal mouse anti-c Myc antibody, 1:200 (Cell Signal-
ing, 2276); biotinylated anti-rabbit IgG and biotinylated anti-mouse IgG, 1:200
A A M A E V Q L L E S G G G L V Q P G G S L R L S C A A S X X X X X X X X X X W V R Q A P G
CDR-H1
K G L E W V S X X X X X X X X X X X X X X X X X X X T I S R D N S K N T L Y L Q M N S L R
CDR-H2
A E D T A V Y Y C A X X X X X X D Y W G Q G T L V T V S S G G G G S G G G G S G G G G S T
CDR-H3 Linker
P S D I Q M T Q S S L S A S V G D R V T I T C X X X X X X X X X X X W Y Q Q K P G K A P K L
CDR-L1
L I Y X X X X X X X G V P S R F S G S G S G T D F T L T I S S L Q P E D F A T Y Y C X X X X X
CDR-L2 CDR-L3
X X X X F G Q G T K V E I K R A A A H H H H H H G A A E Q K L I S E E D L N
His-Tag
B
CDR-H1:
ISPC 1 G F T F S S Y A M S
ISPC 2 G F T F S S Y A M S
ISPC 3 G F T F S S Y A M S
ISPC 4 G F T F S S Y A M S
ISPC 5 G F T F S S Y A M S
ISPC 6 G F T F S S Y A M S
CDR-H2:
ISPC 1 S I T A E G T H T W Y A D S V K G R F
ISPC 2 R I K I F G S K T K F A D S V K G R F
ISPC 3 R I S V A G R R T A Y A D S V K G R F
ISPC 4 P I A S R G A R T N Y A D S V K G R F
ISPC 5 S I H P K G Y P T R Y A D S V K G R F
ISPC 6 R I Q F F G S H T Y F A D S V K G R F
CDR-H3:
ISPC 1 K T S YR F
ISPC 2 K H S TH F
ISPC 3 K K RPP F
ISPC 4 K K P SS F
ISPC 5 K S T TP F
ISPC 6 K H STH F
C
CDR-L1:
ISPC 1 R A S Q S I S S Y L N
ISPC 2 R A S Q S I S S Y L N
ISPC 3 R A S Q S I S S Y L N
ISPC 4 R A S Q S I S S Y L N
ISPC 5 R A S Q S I S S Y L N
ISPC 6 R A S Q S I S S Y L N
CDR-L2:
ISPC 1 K A S R L Q S
ISPC 2 R A S S L Q S
ISPC 3 A A S S L Q S
ISPC 4 K A S P L Q S
ISPC 5 A A S S L Q S
ISPC 6 R A S I L Q S
CDR-L3:
ISPC 1 Q Q K W D P P R T
ISPC 2 Q Q L Q S T P R T
ISPC 3 Q Q M G R D P R T
ISPC 4 Q Q S M Q V P S T
ISPC 5 Q Q M G R D P R T
ISPC 6 Q Q N R R I P R T
FIG. 1. Shown are the amino acid sequences of the �- and �-cell–specific ISPCs. A: Amino acid sequences of heavy (H, red) and light (L, blue)
chain. Boxes indicate the CDRs. X � variable amino acids within CDRs. B and C: Comparison of amino acids within CDR-H1-CDR-H3 (B) and
within CDR-L1-CDR-L3 (C) (variable amino acids are marked in green).
S. UEBERBERG AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 58, OCTOBER 2009 2325

(Linaris, BA-1000, BA-2001); Cy3-conjugated goat anti-mouse IgG, 1:200 (Jack-
son ImmunoResearch Laboratories, 115-165-044); Cy3-conjugated goat anti-
guinea pig IgG, 1:800 (Jackson ImmunoResearch Laboratories, 106-165-003).
Third reagents were Cy2-conjugated streptavidin, 1:200 (Jackson ImmunoRe-
search Laboratories, 016-220-084). Tissue slides were analyzed using a Zeiss
Axioplan microscope.
Electron microscopy. Rats were transcardially perfused with PBS (2.5%
glutaraldehyde) before pancreas extraction, postfixed in 2.5% (wt/vol) glutar-
aldehyde, rinsed with PBS, postfixed in 1% osmiumtetroxide, dehydrated in
ascending concentrations of ethanol and propylenoxide, and embedded in
durcupan. Ultrathin sections were incubated with the anti-c Myc antibody
(1:100; Cell Signaling, 2276) and a biotinylated anti-mouse IgG, 1:200 (Linaris,
BA-2001). Finally, all sections were stained with gold-labeled streptavidin
particles (1/20, 10 nm; Aurion, Wageningen, the Netherlands). After rinses,
sections were postfixed with 2.0% (wt/vol) glutaraldehyde for 5 min and
counterstained. Sections were analyzed on a Zeiss 109 transmission electron
microscope.
Labeling SCAs with 125I. The SCAs (0.1 mg) were labeled with 125I using the
chloramine-T method as described previously (23).
Cell culture. INS-1 (kind gift from C. Wollheim, Geneva), AR42J (ATCC,
Manassas, VA), and �-TC1 cells (ATCC, Manassas, VA) were grown (23,24)
and processed as described (23).
Radioactive in vitro assay. The assay was performed as described previ-
ously (23), except that an electronic pulse area analysis (CASY Technology,
Reutlingen, Germany) was used to estimate the average cell volume (fl). To
evaluate specificity of binding, SCAs were preincubated with selected unla-
beled SCAs (20 �g) for competition assays.
Pharmacokinetic analysis. Rats injected with 100 �g radiolabeled SCAs
were killed at indicated time points, and blood samples were obtained,
followed by sedimentation of cellular material and precipitation of superna-
tant with trichloroacetic acid. Radioactivity associated with pellets and
supernatant was measured. Blood content of radiolabel was expressed as a
percentage of injected dose per gram of blood (%ID/g).
Intraperitoneal glucose tolerance test. CD-rats received an intraperito-
neal glucose tolerance test (IPGTT) 7 days after intravenous injection of SCAs.
Rats were fasted for 4 h before the experiments. After baseline blood
sampling, animals received an intraperitoneal injection of glucose (2 g/kg
body wt) with glucose, insulin, and glucagon levels measured at 30, 60, and
120 min later. Blood samples were taken from tail vein, and glucose was
determined by a clinical analyzer (Nova Biomedical, Ro¨dermark, Germany),
insulin with ELISA (Mercodia, Uppsala, Sweden), and glucagon with an
enzyme immunosorbent assay (Alpco, Salem, NH).
Cell viability and apoptosis. INS-1 or �-TC1 cells (106) were exposed
overnight to SCAs (5 �g or 20 �g). Apoptosis was assessed with FITC-
Annexin-V/propidium iodide (Pharmigen), and analysis was performed with a
FACSscan flow cytometer. Viability was determined by staining the cells with
calcein-AM (Calbiochem) and propidium iodine (Molecular Probes), photo-
graphed with a fluorescence microscope connected to a digital camera, and
images were digitally processed using Cell P software (Olympus) and Photo-
shop 6.0 software (Adobe) (25). Values were compared with those in
nontreated controls.
Biodistribution of [125I]-SCA B1, IPGTTs, and estimation of �-cell
mass. In this set of experiments, an IPGTT was performed 14 days after
diabetes induction (see above for details of these procedures, except that
insulin and glucagon levels were not determined) in low-dose STZ (n � 5;
fasting plasma glucose [FPG]: 250 � 10 mg/dl), high-dose STZ (n � 5; FPG:
490 � 97 mg/dl), and nondiabetic control rats (n � 7; FPG: 90 � 14 mg/dl). On
the following day, the [125I]-SCA B1 (each animal got 0.002 �Ci/g of body wt)
was injected intravenously in these animals. Two hours after injection, the
animals were killed, and pancreases were removed, weighed, and assayed in
a gamma counter for radioactivity (26). Accumulation of SCA B1 was
expressed as a %ID/g of tissue, corrected for background, and estimated in the
glandula parotis. The �-cell mass of the corresponding pancreases was
estimated by morphometry (27) according to the following formula: �-cell
fraction (%) � insulin-positive area/total pancreatic area; �-cell mass per
pancreas (milligram) � �-cell fraction � pancreatic weight (milligram).
Statistics. Curve fits were modeled in form of nonlinear regression. For
washout experiments, data were adapted to an exponential decay function
with y � a � b*exp( c*x), where x denotes the time axis and y the measured
count rate. For saturation experiments, results were adapted to an exponen-
tial rise function as y � a � b*(1 exp[ c*x]). Decision criteria were squared
regression coefficients (r2). Parametric comparisons of continuous data were
calculated with Student’s t test for unpaired data with unequal variance. Main
null hypothesis was equal distribution of measured counts in both investigated
cell types or with and without preincubation, respectively. Area under the
curve (AUC) for glucose, insulin, and glucagon was calculated using the
A
C
E
G
I
B
D
F
H
J
FIG. 2. Immunofluorescence analyses of binding selectivity to pan-
creatic islets after intravenous application of ISPCs, a control ISPC
without an insert, or SCAs in a rat (n � 4 rats per group, 30–40
sections and 60–80 islets per rat). Double staining of ISPCs (A–F,
green) and insulin (A, C, and E, red) or glucagon (B, D, and F, red)
and nucleus using DAPI (blue). The ISPC1 (A and B) colocalized
exclusively with insulin, whereas the ISPC3 (C and D) colocalized
selectively with glucagon. In contrast, the control ISPC without an
insert (E and F) was not detectable in the islets. In another set of
experiments, double staining of SCAs (G–J, green) and insulin (G
and I, red) or glucagon (H and J, red) and nucleus using DAPI (blue)
was performed. This confirmed the highly selective uptake of SCA
B1 in �-cells (G and H) and of SCA A1 in �-cells (I and J). All images
were acquired at magnification �40. (A high-quality digital repre-
sentation of this figure is available in the online issue.)
IMAGING AGENTS SELECTIVE TO PANCREATIC �-CELLS
2326 DIABETES, VOL. 58, OCTOBER 2009 diabetes.diabetesjournals.org

trapezoidal method. All calculations have been performed with KaleidaGraph
4.0.3 for Macintosh Computers (Synergy Software, Reading, PA).
RESULTS
Generation of SCAs binding selectively to either �-
or �-cells in rats. To generate agents specifically binding
to pancreatic islets, a phage library was screened for SCAs
on rat islets using two different approaches: A: The library
was injected into rats in vivo, and islets were isolated after
a circulation time of 5 min. B: Pancreatic islets were
directly isolated, and the library was panned in the islets in
vitro. After five rounds of selection, a marked increase in
the phage transducing units per islet over successive
rounds of panning was found, demonstrating a 700- and
500-fold enrichment with approach A and B, respectively.
Subsequently, the DNA encoding the corresponding
phage-displayed SCAs was sequenced. By these means,
four islet-specific phage clones (termed ISPC1 � SCA B1,
ISPC2 � SCA B2, ISPC3 � SCA A1, ISPC4 � SCA A2) were
identified by approach A (Fig. 1). Approach B also yielded
four ISPCs, two of which were identical to those derived
from approach A (ISPC1 and 2). The other two ISPCs were
termed ISPC5 and 6 (SCA B3 and SCA B4).
Binding selectivity and subcellular localization of
SCAs. To determine the binding selectivity of the ISPCs
to rat islets in vivo, the ISPCs were injected intrave-
nously and allowed to circulate for 2 h. Subsequently,
the animals were killed and the pancreas and control
organs were harvested and prepared for immunohisto-
chemical analyses. Immunostaining for ISPC1 was
readily detectable in the cytoplasm of islet cells, where
it was colocalized to insulin, but not glucagon staining,
liver kidney spleen muscle lung
ISPC1
ISPC3
control
SCA B1
SCA A1
FIG. 3. Immunofluorescence analyses of binding to control tissue after intravenous application of ISPCs, a control ISPC without an insert, or
SCAs in a rat (n � 4 rats per group, 30–40 sections per rat). Staining in green color for ISPCs or SCAs. Nuclei were stained with DAPI in blue
color. Neither the IPSCs nor SCAs revealed any binding activity to the tested control organs. All images were acquired at magnification �40. (A
high-quality digital representation of this figure is available in the online issue.)
FIG. 4. Ultrastructural analyses of exact intracellular localization of the
SCAs after intravenous application in a rat. Transmission electron micros-
copy detected the �-cell–specific SCA B1 (A and B) and the �-cell–specific
SCA A1 (C and D) in the endoplasmatic reticulum (A and C) and at the
secretory granule membrane (B and D) of the respective target cells exclu-
sively (n � 4 rats per group, 20–30 sections per rat). Scale bars, 90 nm.
S. UEBERBERG AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 58, OCTOBER 2009 2327

suggesting selective binding to �-cells (Fig. 2A and B).
Similar staining patterns were found for ISPC2, 5, and 6
(data not shown). In contrast, ISPC3 (Fig. 2C and D) and
4 (data not show) were found to be exclusively colocal-
ized with glucagon staining, indicating that these ISPCs
were selectively binding to islet �-cells. Of note, no
binding to exocrine cells (Fig. 2A–D) and several other
control organs (liver, kidney, spleen, heart, and lung)
was detectable in any of the ISPCs (Fig. 3). Further-
more, control experiments using an ISPC without the
insert did not reveal any binding activity to pancreatic
islets or other structures (Fig. 2E and F).
Subsequently, soluble SCAs containing a c-Myc tag and
a His6 tag were generated from all six ISPCs in small-scale
cultures. These SCAs were then purified by metal affinity
chromatography and administered intravenously into rats.
These experiments confirmed the highly selective cyto-
plasmatic uptake of SCA B1 (Fig. 2G and H) and B2–4
(data not shown) in �-cells and of SCA A1 (Fig. 2I and J)
and A2 (data not shown) in islet �-cells, whereas no
-2
0
2
4
6
8
10
12
14
Ins1 α-TC1 AR42J Ins1 α-TC1 AR42J
C
A
R
(c
pm
/fl
)
A
C
B
*
-1
0
1
2
3
4
5
6
7
C
A
R
(c
pm
/fl
)
*
4
6
8
10
12
14
0 20 40 60 80 100
C
A
R
(c
pm
/fl
)
Time (minutes)
4
6
8
10
12
14
0 20 40 60 80 100
C
A
R
(c
pm
/fl
)
Time (minutes)
D
FIG. 5. Analyses of the binding process of selected [125I]-labeled SCAs to different endocrine and exocrine cell lines in vitro and determination
of their pharmacokinetic profiles in vivo. Binding specificity of SCA B1 to INS-1 cells (A, *P � 0.0016 vs. �-TC1 or AR42J) and SCA A1 to �-TC1
cells (B, *P < 0.0001 vs. INS-1 or AR42J). Time course of binding of SCA B1 to INS-1 cells (C, t1/2 � 8.0 min) or SCA A1 to �-TC1 cells (D, t1/2 �
5.3 min). Competition assay of SCA B1 (E, *P < 0.0001 vs. preincubation (PI) with SCA B1) or SCA A1 (F, *P < 0.0001 vs. preincubation with
SCA A1) with unlabeled SCAs. Dose response of SCA B1 binding to INS-1 cells (G, r2 � 0.96) or SCA A1 to �-TC1 cells (H, r2 � 0.96). Time course
of elimination of SCA B1 (I, t1/2 � 22.7 min, r
2
� 0.87) or SCA A1 (J, t1/2 � 19.2 min, r
2
� 0.97) from the vascular system. Error bars represent SEM.
CAR, cell-associated radioactivity.
IMAGING AGENTS SELECTIVE TO PANCREATIC �-CELLS
2328 DIABETES, VOL. 58, OCTOBER 2009 diabetes.diabetesjournals.org