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Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Universidad Autonoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain
Address all correspondence and requests for reprints to: Dr. Santos Mañes, Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Universidad Autonoma, Campus de Cantoblanco, E-28049, Madrid, Spain. E-mail: smanes{at}samba.cnb.uam.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been demonstrated recently that IGF-I, originally considered be
a systemic regulator of growth, may play a critical role in cell
proliferation control (5). IGF-I exerts its effects through interaction
with the type-1 IGF receptor (IGF-1R), a heterotetrameric protein
composed of two 
and two ß subunits linked by disulphide bridges
(6). IGF-I also binds with 100x less affinity to the homologous
insulin receptor and to the type-2 IGF receptor with even lower
affinity (7). In addition, both IGF-I and IGF-II, but not insulin, bind
to specific, soluble binding proteins present in extracellular fluids
(8, 9). Six specific IGF-binding proteins (IGFBP) have been cloned and
characterized to date, but their biological role is far from being
understood (5). Evidence suggests that IGFBP act as carrier, increasing
the half-life of IGF in serum (10) and transporting IGF through the
vasculature and across capillary surfaces to cellular sites (11).
Finally, IGFBP may inhibit (12, 13, 14) and/or enhance (14, 15, 16) IGF
activity.
In cells, IGF-I mediates either short-term, insulin-like effects, which include metabolic effects such as stimulation of glucose uptake, glycogen, and lipid synthesis, or long-term mitogenic effects such as stimulation of protein, RNA, and DNA synthesis, as well as cell proliferation (5). IGF-I promotes mesoderm, adipocyte, neuron, oligodendrocyte, ovary, and testicular cell differentiation. It also has been implicated in hematopoiesis, and IGF-1R are expressed in hematopoietic cells such as human monocytes, B lymphocytes, and peripheral blood lymphocytes (PBL) (17, 18). In vitro, IGF-I blocks IL-3 deprivation-induced apoptosis in two murine hematopoietic cell lines, Ba/F3 and FDCP-Mix, as it does in primary cultures of IL-3-dependent mouse bone marrow-derived mast cells (19). In combination with IL-7, IGF-I seems to regulate pro-B cell differentiation of B220- bone marrow cells in short-term cultures (3).
Monoclonal antibodies (mAb) of defined specificity might be used as a complementary approach to study the structure-function relationships of IGF-I (20). These mAb can be used to gain insight into the topological features of the IGF-I surface and the IGF-I domains that remain exposed after interaction with IGFBP and receptors. Indeed, mAb to IGF-I have been used both to inhibit and to increase the biological activity of IGF in vitro and in vivo (21, 22). We have characterized a large panel of mAb to various IGF-I epitopes, which were generated for blocking or enhancing IGF-I biological activity. We describe the functional epitope mapping of IGF-I and its role on IGF-I binding to IGFBP and the IGF-1R. Finally, some of these mAb also block the IGF-I-induced in vitro survival of a pro-B cell line and thus may be useful as IGF-I antagonists.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell growth assays were performed under serum-free conditions. Ba/F3 cells (2 x 104/well) were incubated in 96-well plates in RPMI-1640 containing 2 mM glutamine, 1 mM sodium pyruvate, 0.5% BSA RIA Grade (Sigma, St. Louis, MO). The mAb and recombinant human IGF-I (rhIGF-I) were coincubated 2 h at 4 C before being added to the cells. After 20 h, cells were pulsed for 4 h with 0.5 µCi/well of [3H]TdR (Amersham, Aylesburg, UK), and nuclei were harvested on glass fiber filters using a cell harvester (LKB-Wallac, Uppsala, Sweden). DNA [3H]TdR incorporation was determined with a liquid scintillation counter. The results are the mean and SD of three independent experiments.
For cell cycle analyses, Ba/F3 cells were incubated in 24-well plates, as described above. After 20 h incubation, cells were centrifuged, resuspended in 100 µl PBS, and stained with propidium iodide using the DNA-Prep Stain kit (Coulter Corp., Hialeah, FL). Cell cycle analysis was carried out in a flow cytometer equipped with a pulse processing facility to enable the discrimination of cell doublets (Epics XL, Coulter).
Immunogens
rhIGF-I, rhIGF-II, glycosylated rhIGF-I (25), and a semipure
preparation of rhIGF-I (IGF-IG50) were provided by the Peptide Hormones
Division, Pharmacia (Stockholm, Sweden) and used as immunogens both in
free form and coupled to keyhole limpet hemocyanin (KLH) by the
two-step glutaraldehyde method (26). SAPV8 IGF-I(4+9), an IGF-I
fragment obtained by proteolytic cleavage with the Staphylococcus
aureus V8 protease, and which contains the amino acid sequence
IGF-I[13–20]-[59–70] linked by a disulphide bridge
(Pharmacia), was coupled to KLH in a one-step glutaraldehyde
method.
Peptides IGF-I[1–13] and IGF-I[18–30], both corresponding to the IGF-I B-domain; IGF-I[29–41], representing the IGF-I C-domain; and IGF-I[55–70], spanning the C-terminal part of A-domain and the complete IGF-I D-domain, were synthesized in solid phase using standard Fmoc chemistry (27) in a Multiple Peptide Synthesizer (Abimed, Langefeld, Germany) and KLH-coupled using either the two-step glutaraldehyde method or the heterobifunctional reagent 4-(maleidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester, basically as described (26). These four peptides were chosen for immunization because they cover approximately the total IGF-I sequence.
mAb production
Eight-week-old BALB/c, C3H/He, C57BL/10, and Biozzi mice
received sc injections of 30–60 µg immunogen in Freund’s complete
adjuvant and were boosted sc at 28 and 56 days in incomplete adjuvant.
Serum was sampled at days 37 and 65 to test the polyclonal anti-IGF-I
response, and the mice received an additional ip boost 1 month later.
Animals selected for fusion received 40–60 µg immunogen ip and the
same dose iv at days -3 and -2 before cell fusion. Spleen and lymph
node lymphocytes were fused with the murine plasmacytoma P3X63-Ag8.653,
essentially as described (28). Hybridomas were selected in RPMI-1640
medium supplemented with 10% FCS, 2 mM
L-glutamine, 1 mM sodium pyruvate,
0.1 mM hypoxanthine and 10 µM azaserine. Two
weeks after fusion, supernatants from growing wells were screened for
anti-IGF-I activity in enzyme-linked immunosorbent assay (ELISA) and
RIA, as described below. Selected antigen-specific antibody-secreting
hybrids were stabilized by limiting dilution in two cloning steps.
Monoclonal antibodies (mAb) were produced either in culture medium or
in ascites fluid. Ascites tumors were established by ip injection of
1–1.5 x 106 cells in Pristane-primed BALB/c or
(BALB/c x C57BL/10) F1 mice. The mAb were partially
purified from ascites fluid by sequential precipitation with caprylic
acid and ammonium sulphate (29) or from tissue culture medium by
affinity chromatography with protein-A Sepharose (Pharmacia).
Antibodies were biotinylated as previously reported (30).
Hybridoma and serum screening assays
Antibodies in serum and in hybridoma culture supernatants were
screened using both an antigen-coated plate ELISA and a liquid-phase
RIA. In ELISA, microtiter plates were coated with rhIGF-I at 1 µg/ml
overnight at 4 C and, after blocking with 0.5% BSA in PBS, tissue
culture supernatants or serum dilutions were added. After incubation at
37 C, the reaction was visualized using a peroxidase-labeled goat
antimouse Ig antiserum (GAM-PO; Tago Inc., Burlingame, CA), followed by
o-phenylenediamine (OPD; Sigma) and H2O2. In
RIA, tissue culture supernatant or serum dilutions were incubated with
125I-IGF-I (150–200 µCi/mg) in a final volume of 400
µl PBS buffer containing 10 mM ethylenediaminetetraacetic
acid, 0.01% NaN3, 0.25% BSA, and 0.5% normal mouse
serum. After 16 h at room temperature, bound radioactivity was
precipitated by adding GAM and 15% wt/vol PEG 6000, followed by 30 min
centrifugation at 1,500 x g.
mAb reactivity to rhIGF-II and human insulin (Sigma), as well as apparent affinity constants, were determined in a competitive RIA, basically as described above, followed by the Scatchard transformation of the data.
Epitope mapping
Antibodies were studied in three simultaneous binding
assays. 1) Sandwich assay. The mAb were immobilized on microtiter
plates. After blocking with PBS-0.5% BSA, rhIGF-I (1–5 µg/ml) or an
irrelevant protein were added, followed by a second biotinylated
anti-IGF-I mAb. The reaction was developed using peroxidase-labeled
streptavidin (Strep-PO; Sigma) followed by OPD and
H2O2. A mAb pair was considered to bind
simultaneously to IGF-I when a greater than 5-fold optical density
increase was observed in the presence of IGF-I compared with the same
pair in the absence of growth factor. 2) Competitive assay. Plates
were coated with rhIGF-I at 0.3–0.9 µg/ml overnight at 4 C.
Previously-tested biotinylated mAb mixed with different concentrations
of each unlabeled mAb (0–150 µg/ml) were then added and the reaction
developed, using Strep-PO, as above. Two mAb compete for binding to
IGF-I when a signal decrease greater than 30% is observed. 3)
Additive assay. rhIGF-I (10 µg/ml in 10 mM acetate
buffer, pH 4.5) was covalently coupled to an activated
carboxymethylated dextran CM-5 sensor chip (Pharmacia Biosensor,
Sweden) through amino groups, as described (31). Each two-site binding
assay cycle was performed by injecting the first mAb at 50 µg/ml in
HEPES-buffered saline plus 0.05% P-20 for 6 min, followed by 1 min
injection of the second mAb at the same concentration in continuous
flow at 5 µl/min. Under these conditions, saturation of the epitope
recognized by the first mAb was assured because reinjection of the same
mAb resulted in no additional binding. The antibodies were removed
subsequently with a 1-min pulse of 100 mM HCl at the same
flow rate. mAb binding was quantified as the change measured over
baseline signal (in resonance units) 30 sec after the end of each mAb
pulse. Two mAb are considered to bind IGF-I simultaneously when an
increase of at least 70% takes place after injection of the second
mAb.
Multisite binding experiments were performed by sequential injection of four different anti-IGF-I mAb before the HCl pulse. To assure saturation of the epitope recognized by each secondary mAb, concentrations and injection times required were tested by two-site binding.
IGF-I/IGFBP complex detection assays
Binding of mAb to the IGF-I/IGFBP-1 or -3 complexes was measured
in a solid-phase ELISA, essentially as described above. Human IGFBP-1
and -3 were immobilized at 1 and 0.5 µg/ml, respectively. After
blocking, rhIGF-I or BSA were added and incubated for 1 h at room
temperature. The mAb were added and the reaction developed with GAM-PO
and OPD.
In the competition assay, either IGFBP-1 or -3 was incubated with 125I-IGF-I for 12 h at room temperature. Selected mAb from each epitopic cluster were added at a concentration able to bind 50% of the tracer. After 2 h incubation, mAb-bound radioactivity was precipitated as described above.
IGF-I receptor quantification
Ba/F3 cells (2 x 106) or 3T3-IGF-IR cells at
75–90% of confluence in 24-well plates were exposed to
125I-IGF-I together with varying concentrations of
unlabeled rhIGF-I, rhIGF-II, or insulin. The cells were incubated at 4
C for 6 h, washed with cold buffer, and counted. Fibroblasts were
solubilized with 2% SDS in PBS before counting. The radioactivity
bound to the cells at 100 nM unlabeled rhIGF-I was
considered nonspecific binding and subtracted from each data point. The
Scatchard transformation was used to calculate the apparent affinity
constant and the site number per cell.
mAb recognition of IGF-I bound to IGF-1R
Ba/F3 cells (2 x 105 cells/ml) were incubated
with 2–5 µg/ml of rhIGF-I in PBS plus 0.5% BSA, 1% FCS, and 0.01%
NaN3 for 1 h at 4 C. Biotinylated mAb diluted in the
same buffer were added, followed by phycoerythrin-labeled avidin
(Avidin-PE, Southern Biotechnologies, Birmingham, AL). Cell-associated
fluorescence was visualized by flow cytometry. In some experiments,
biotinylated mAb were incubated 2 h with 10 µg/ml rhIGF-I or
IGF-I[55–70] synthetic peptide before the addition to the cells.
For competition experiments, biotinylated rhIGF-I preincubated with
unlabeled mAb (1–10 µg) was added to Ba/F3 cells. After 1 h
incubation, avidin-PE was added and analyzed as above. A similar
protocol was followed for BALB/c 3T3-IGF-IR cells, but, in this case,
125I-IGF-I was used. The radioactivity bound to the cells
was solubilized by addition of 2% SDS in PBS and counted in a 
counter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice with either high antibody titers or particular recognition patterns were selected for cell fusions. In spite of the high serum titers obtained against IGF-I, the low percentage of specific hybridomas obtained from fusions of recombinant or mutant IGF-I-immunized mice was surprising. However, in fusions from mice immunized with synthetic peptides, a clearer relationship between antiserum titer and the number of specific hybrids was observed.
More than 140 anti-IGF-I antibodies were obtained and characterized for
their reactivity to IGF-I and IGF-II using antigen capture and
solid-phase bound antigen ELISA (data not shown). On the basis of their
reactivity patterns, 28 hybrids from 10 different fusions were selected
(Table 1
). These mAb have apparent affinity constants
for IGF-I ranging from less than 106 to 1010
M-1, and their crossreactivity with IGF-II,
studied in competitive binding assays, ranged from less than 0.1% to
800%. None recognized human insulin in solid phase-bound antigen ELISA
(data not shown) or competitive RIA (Table 1). Antibodies are of IgG1,
IgG2a, IgG2b, and IgM isotypes (Table 1); all express kappa light
chain.
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) based on their ability either to bind simultaneously or to
compete with one another for IGF-I binding. Three different assays were
performed including antigen sandwich ELISA, competitive ELISA, and an
additive assay in biospecific interaction analysis (BIAcore). These
assays provide information on mAb pairs that bind to the same IGF-I
molecule, mAb pairs that bind to overlapping or identical epitopes on
solid-phase immobilized IGF-I, and mAb pairs showing an interfering or
noninterfering interaction with immobilized IGF-I, respectively. All
mAb mapping to a given epitopic cluster inhibit the binding of the
other mAb in that cluster to immobilized IGF-I in the competitive and
additive assays (Fig. 1). Same-cluster mAb do not bind
simultaneously to IGF-I in the sandwich assay, suggesting that there
are no two identical epitopes on the IGF-I molecule.
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). That is, mAb which can bind simultaneously in the
sandwich assay compete neither in the competitive nor in the additive
assays, and vice versa. The only exceptions observed are
cluster VII (BB11C11) and cluster I (BW1C1, BW1C8, BW4H10, BW5G5) mAb.
They bind simultaneously in the sandwich assay but compete in both the
competitive and additive assays when cluster I mAb are added as first
antibody. This asymmetry is observed only when the antigen is bound in
solid phase, suggesting that cluster I antibody binding to IGF-I in
solid-phase induces a conformational change that modifies the epitope
recognized by mAb BB11C11 (specific for cluster VII). The pair-wise
epitope mapping was confirmed by sequential binding of the anti-IGF-I
antibodies in BIAcore. As shown in Fig. 2, mAb specific
for clusters I, II, III, and IV do not interfere with one another in
their binding to IGF-I, suggesting that four different mAb,
representing independent epitopic clusters, can bind simultaneously to
IGF-I.
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. Antibodies specific for epitope
clusters II, V, and VII involve principally residues in the B-domain;
clusters III and IV map to amino acid sequences IGF-I[60–70] and
IGF-I[1–13], respectively. Cluster VI probably includes the first
-helix of the A-domain and, at least for BW1E7 and GG6C1, the
N-terminal part of the B-domain; cluster VIII specific mAb involves
mainly the C-terminal of the B-domain. There is no conclusive data that
precisely define the epitope recognized by epitopic cluster I-specific
mAb, but the decreased reactivity with the mismatched IGF-I indicates
that they must recognize an epitope closely related to that of cluster
VI mAb. In addition, their low crossreactivity with hIGF-II, similar to
that reported for antibodies directed against the IGF-I-C-domain (32),
as well as with cIGF-I, suggest that at least a part of the epitope
is located in the boundary between the C- and A-domains.
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).
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). Cluster IV mAb
were not tested in this assay because they immunoprecipitate
125I-IGF-I poorly.
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In a first attempt to define the IGF-I epitopes that remain exposed
after interaction with the receptor, we analyzed mAb binding to Ba/F3
or 3T3-IGF-IR cells alone or preincubated with IGF-I. Only the cluster
III mAb recognize IGF-I, detected as an increase in fluorescence
intensity when Ba/F3 cells were preincubated with IGF-I (Fig. 6). None of the mAb specific for any other cluster bind
differentially to these cells in the presence or absence of IGF-I. The
specificity of cluster III mAb binding for Ba/F3 cells was demonstrated
by preincubation of these mAb with a molar excess of IGF-I or the
IGF-I(55–70) synthetic peptide. As expected, this treatment reduced
the cell-associated fluorescence intensity. Similar results were
obtained using 3T3-IGF-IR cells (data not shown).
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), suggesting an overlapping
of the epitopes recognized by these mAb and the IGF-I receptor. The
only exception were the mAb with an apparent Ka for IGF-I
lower than 1 µM-1, including those that
recognize cluster IV (OD3G6 and OD6E11), probably caused by their low
affinity. Surprisingly, a slight inhibition of IGF-I binding to the
cells also was observed in the presence of cluster III-specific mAb,
although they recognized IGF-I once it was bound to the receptor.
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) and promotes
cell growth, although to a lesser extent than IL-3 (data not shown).
Furthermore, even at saturating IGF-I concentrations, approximately
20% of Ba/F3 cells undergo apoptosis (Fig. 8). As expected, some
anti-IGF-I mAb prevent IGF-I-promoted survival (Fig. 9)
and block IGF-I-induced proliferation (Table 2) of Ba/F3
cells in a dose-dependent fashion. In agreement with the binding
inhibition experiments, the effect of mAb was dependent on their
affinity for IGF-I. The cluster III mAb showed less inhibitory effect
than mAb for other domains with similar apparent affinity constants. In
addition, the presence of recombinant nonphosphorylated human IGFBP-1,
used here as a control, also blocks cell survival and promotes
apoptosis, similar to anti-IGF-I mAb.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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.
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The mAb collection has been used to characterize the IGF-I domains involved in its interaction with IGFBP-1 and -3. Of the anti-IGF-I mAb described here, only those specific for cluster III, directed to the D-domain, bind to IGF-I when it is complexed with one of the binding proteins. The epitope recognized by these mAb seems unaltered after interaction with the IGFBP, as the titration curves obtained against IGF-I, alone or complexed, show similar slopes (data not shown). This suggests that the IGF-I carboxy-terminal D-domain does not participate in the interaction with either IGFBP-1 or -3 and agrees with the mutagenesis studies in which D-domain deletion has little or no influence on the interaction of IGF-I with IGFBP-1, IGFBP-2, and IGFBP-3 (39). Because accumulated evidence (40) also shows reduced affinity of the [1–62]IGF-I mutant for IGFBP-4 and IGFBP-5 compared with IGF-I, it could be interesting to analyze whether these mAb bind to IGF-I complexed to these IGFBP.
The cluster III mAb also recognize IGF-I bound to the IGF-1R expressed on Ba/F3 or to 3T3-IGF-IR cells. This recognition is specific, because it is blocked when the mAb are preincubated with IGF-I or with the IGF-I[55–70] synthetic peptide. These results suggest that the IGF-I D-domain remains exposed after its interaction with the IGF-1R and supports the mutagenesis studies in which the [1–62]IGF-I mutant shows only marginally decreased affinity to IGF-I receptor binding (41). In competition experiments, cluster III mAb showed unexpected partial inhibition of IGF-I binding to both cell lines. Nuclear magnetic resonance studies indicate that the IGF-I core is very similar to that of insulin, whereas the C- and D-domains are poorly resolved in these structures because of their intrinsic mobility (42). Several authors have modeled these domains as flexible flaps flanking the receptor-binding cleft motif. Synthetic analogues of the IGF-I D-domain block IGF-I receptor autophosphorylation and inhibit IGF-I-induced proliferation in several prostatic cell lines, suggesting that the D-domain binds to the IGF-1R (43). The binding inhibition of these mAb thus could be caused by the hiding of some residues involved in the interaction with the receptor. We prefer, however, the explanation that mAb binding to cluster III decreases D-domain flexibility, hindering the interaction between the receptor and the binding cleft motif. Other possibilities cannot be ruled out, such as direct contact between the D-domain and the receptor as a first step in IGF-I binding.
IGF-I residues that interact with IGFBP and IGF receptors have been identified, mostly by homologous scanning mutagenesis (39, 41). These are residues 1–3 and 49–51 for the binding proteins and residues 21, 23, 24, 44 and tyrosines 31 and 60 for the receptor. Extrapolation of these amino acids on a partially-resolved three-dimensional structure of IGF-I (42) indicates that the IGFBP and the IGF-1R binding surfaces are on opposite sides of the IGF-I molecule. However, some IGFBP inhibit IGF-I biological activity but not that of IGF-I analogs with low affinity for the IGFBP (44). Our data indicate that the epitopes defined by mAb specific for clusters I, II, IV, V, VI, VII, and VIII overlap with those for IGFBP-1 and -3 and, in addition, these mAb inhibit IGF-I binding to the receptor. Because these mAb cover almost the entire IGF-I exposed surface, this suggests that there is an overlap between the IGF-1R and the IGFBP epitopes and may explain the inability to date to obtain IGFBP/IGF-I/IGF-1R tertiary complexes. Furthermore, preincubation of IGF-I with nonphosphorylated E. coli-produced hIGFBP-1 inhibits IGF-I-promoted survival of Ba/F3 cells at doses comparable with those of anti-IGF-I mAb with similar affinity for this growth factor, even when the unphosphorylated form of IGFBP-1 has been shown to enhance IGF-I biological activity in several cell lines (45).
Our experiments confirm the observation that IGF-I promotes Ba/F3 cell survival in the absence of IL-3, as well as increasing [3H]-thymidine incorporation into the DNA of these cells. However, IGF-I promotes only a slight increase in cell number after 5 days of in vitro culture as compared with the IL-3-induced proliferation. The lower IGF-I-induced proliferative response may reflect differences in intracellular signaling or a low level of IGF-1R expression on these cells. Both induction of DNA synthesis and suppression of apoptotic death may be mediated by independent signaling pathways, as recently suggested for IL-3 (46). These effects can be inhibited by the anti-IGF-I mAb, which block IGF-I binding to IGF-1R.
IGF-I protects cells from c-myc-induced apoptosis (47), and IGF-1R overexpression promotes ligand-dependent cell transformation (48). Transfection of embryo fibroblasts carrying the null mutation for the IGF-1R with the viral oncogene SV40 T antigen alone (49) or in conjunction with activated Ha-ras (50) does not promote cellular transformation. This suggests that the IGF-1R is necessary for transformation. Indeed, when the receptor number is decreased by specific antisense oligonucleotides, several human and murine tumor cells undergo apoptosis in vivo and decrease their tumor growth rate in syngeneic or nude mice (49, 51). Interestingly, SV40 T antigen expression markedly increases IGF-I secretion (52). Thus, it seems that there is an autocrine loop mediated by the IGF-I/IGF-1R interaction that participates in transformed cell growth.
In summary, we have developed 28 anti-IGF-I mAb that, by simultaneous pair-wise analysis, define 8 epitopic clusters covering almost the entire IGF-I solvent-exposed surface. The binding of cluster III-specific mAb to IGF-I previously complexed with either IGFBP or IGF-1R indicates that the IGF-I D-domain remains exposed after this interaction. The fact that mAb specific for other clusters interfere in the binding of both IGFBP and IGF-1R suggests an overlapping between the IGF-1R and the IGFBP surfaces and, thus, the inability to obtain tertiary complexes. We found that these anti-IGF-I mAb block IGF-I-induced Ba/F3 cell survival, including those specific for cluster III that recognize the IGF-I/IGF-1R complex. This suggests the importance of D-domain flexibility in the ligand-receptor interaction. The availability of an extensive panel of anti-IGF-I mAb directed to epitopes that remain exposed after IGFBP or IGF-1R interaction makes possible their use to interfere with the IGF-I autocrine loop, leading to potential therapeutical applications.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Biotechnology, Fundación Club
de Inversores, E-03001 Alicante, Spain.
Received August 21, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, Those
mAb pairs able to bind to the same IGF-I molecule (noninterfering
interactions); 
, the mAb pairs recognizing proximal or identical
epitopes (interfering interactions); 
, a contradiction in the
results between the two directions of the assay. The mAb BA5F11,
BB11D3, and NL2A4 did not recognize IGF-I when bound to the sensor chip
in BIAcore (
).
