Epithelial stem cells of the eye surface.

OBJECTIVES: Epithelial stem cells of the eye surface, of the cornea and of the conjunctiva, have the ability to give rise to self renewal and progeny production of differentiated cells with no apparent limit. The two epithelia are separated from each other by the transition zone of the limbus. The mechanisms adopted by stem cells of the two epithelia to accomplish their different characteristics, and how their survival, replacement and unequal division that generates differentiated progeny formation are controlled, are complex and still poorly understood. They can be learned only by understanding how stem cells/progenitors are regulated by their neighbouring cells, that may themselves be differently unspecialised, forming particular microenvironments, known as 'niches'. Stem cells operate by signals and a variety of intercellular interactions and extracellular substrates with adjacent cells in the niche. Technical advances are now making it possible to identify zones in the corneal limbus and conjunctiva that can house stem cells, to isolate and expand them ex vivo and to control their behaviour creating optimal niche conditions. With improvements in biotechnology, regenerative cornea and conjunctiva transplantation using adult epithelial stem cells becomes now a reality. RESULTS AND CONCLUSIONS: Here we review our current understanding of stem cell niches and illustrate recent significant progress for identification and characterization of adult epithelial stem cells/progenitors at cellular, molecular and mechanistic levels, improvement in cell culture techniques for their selective expansion ex vivo and prospects for a variety of therapeutic applications.

Expansion of human mesothelial progenitor cells in a longterm three-dimensional organotypic culture of Processus vaginalis peritonei.

A 3D culture system was used to investigate the behaviour of mesothelial cells present in the wall of human processus vaginalis peritonei. Small tissue fragments placed on collagen sponges were cultured for 7, 14 and 21 days in medium supplemented with 10% FBS, and analysed for the expression and distribution of cytokeratins (CKAE1-AE3, CK19), p63, Ki-67, vimentin, CD34, and HBME-1. Before culture, flat mesothelial cells displayed immunoreactivity for cytokeratins, vimentin and HBME-1, while p63 and CD34 were negative. Mesenchymal cells within the stroma were vimentin-positive and endothelial cells of small vessels displayed positive staining for CD34. Cytokeratins, p63 and HBME-1 were negative in all stromal cells. In cultured fragments, flat mesothelial cells positive for vimentin, cytokeratins and HBME-1 proliferated, lining the fragment surface and migrating into the sponge. Capillaries showed morphological alterations; however, their immunoreactivity was comparable with the stroma prior to culture. Cells that had migrated into the sponge and displayed characteristics of mesothelial progenitors, predominantly spindleshaped and stellate, showed heterogeneous expression of markers especially in late phases of cultivation. These cells were constantly positive for vimentin, a small fraction was cytokeratin-positive and a few displayed HBME-1 immunoreactivity. CD34 was found in cells forming small cavities into the matrix, resembling newly formed blood vessels. Cells that had migrated into the sponge could be isolated and expanded in coculture with feeder NIH.3T3 fibroblasts. This system is suitable for studying growth and behaviour of mesothelial cells within their natural environment, providing a good method for isolation and expansion of their progenitor cells.

Primate embryonic stem cells create their own niche while differentiating in three-dimensional culture systems.

Rhesus monkey embryonic stem cells (ESCs) (R366.4), cultured on a three-dimensional (3D) collagen matrix with or without human neonatal foreskin fibroblasts (HPI.1) as feeder cells, or embedded in the collagen matrix, formed complex tubular or spherical gland-like structures and differentiated into phenotypes characteristic of neural, epithelial and endothelial lineages. Here, we analysed the production of endogenous extracellular matrix (ECM) proteins, cell-cell adhesion molecules, cell-surface receptors, lectins and their glycoligands, by differentiating ESCs, forming a micro-environment, a niche, able to positively influence cell behaviour. The expression of some of these molecules was modulated by HPI.1 cells while others were unaffected. We hypothesized that both soluble factors and the niche itself were critical in directing growth and/or differentiation of ESCs in this 3D environment. Creating such an appropriate experimental 3D micro-environment, further modified by ESCs and modulated by exogenous soluble factors, may constitute a template for adequate culture systems in developmental biology studies concerning differentiation of stem cells.

Epitope mapping analysis of apolipoprotein B-100 using a surface plasmon resonance-based biosensor.

Using a surface plasmon resonance (SPR)-based biosensor (BIA-technology), we have studied the interaction of ten different murine monoclonal antibodies (mAbs, all IgG(1)), raised against the main protein constituent of human low density lipoprotein (LDL), i.e. the apolipoprotein B-100 (apoB-100). These mAbs identify distinct domains on apoB-100, relevant to LDL-receptor interaction: epitopes in the amino-terminal region (mAbs L7, L9, L10 and L11: aa 1-1297) and in the middle region (mAb 6B: aa 1480-1693; mAbs 2A, 3B: aa 2152-2377; mAbs 9A, L2 and L4: aa 2657-3248) of native apoB-100. A multisite binding analysis was performed to further characterize the epitopes recognized by all these mAbs. A rabbit anti-mouse IgG(1)-Fc antibody (RAM.Fc) was first coupled to the gold surface in order to capture one anti-human apoB-100 mAb. ApoB-100 protein was subsequently injected and allowed to react with this immobilized, oriented antibody. Multisite binding assays were then performed, by sequentially flowing other mAbs, in different orders, over the sensing surface. The capacity of each mAb to interact with the entrapped apoB-100 in a multimolecular complex was monitored in real time by SPR. The results achieved were comparable to those obtained by western immunoblotting using the same reagents. However, SPR ensures a more detailed epitope identification, demonstrating that BIA-technology can be successfully used for mapping distinct epitopes on apoB-100 protein in solution dispensing with labels and secondary tracers; moreover, compared with conventional immunoassays, it is significantly time saving (CNR-P.F. MADESS 2).

Endothelin-1 in human prostatic carcinoma treated with androgen withdrawal: an immunohistochemical study.

BACKGROUND: Various reports suggest a role for endothelin-1 in prostatic carcinoma. The objective of the current study was to evaluate the changes of the immunodetectable endothelin-1 in prostatic carcinomas characterized by different grades of regression due to total androgen withdrawal. METHODS: An immunohistochemical study was made on eleven prostatic carcinomas treated with neoadjuvant hormonal therapy for 3 months, followed by radical prostatectomy. Another ten specimens of untreated carcinomas were studied for comparison. An appraisal of androgen receptors was associated. A highly specific polyclonal antibody against endothelin-1 and a commercial monoclonal mouse antibody for androgenic receptors were used. RESULTS: In all cases, a prevalent quantity of androgenic receptor-positive tumor cells were present. Neoplastic cells of untreated carcinomas showed a strong and heterogeneous staining for endothelin -1. In unregressed areas of treated cases, the features of endothelin-1 and androgen-receptor staining were the same as those of untreated cases. In areas characterized by moderate histologic regression, the endothelin-1 staining became more heterogeneous. In areas of strong histologic regression, a diffuse membrane staining was often noted. Only in completely regressed cancer cells was a definite loss of immunodetectable endothelin-1 and androgenic receptors observed. CONCLUSIONS: Endothelin-1 is one of the proteins intrinsic to prostatic epithelial cells, both benign and malignant. In cases treated with androgen withdrawal, histologic regression is not uniform. In unmodified areas, immunodetectable endothelin-1 and androgenic receptors also are unmodified, thus suggesting some mechanism that substitutes for the action of androgen. Only neoplastic cells with complete histologic regression also lose androgenic receptors and endothelin-1, whereas the preserved immunostaining in deeply modified prostatic neoplastic cells seems to indicate that these cells still are potentially active.

Naturally-occurring anti-G-CSF antibodies produced by human cord blood B-cell lines infected with Epstein-Barr virus.

INTRODUCTION: Naturally occurring antibodies (auto-Abs) recognizing human granulocyte-colony stimulating factor were detected with high frequency in serum samples obtained from umbilical cord blood of newborns (12 of 65 samples screened) and maternal peripheral blood serum samples from women at the end of gestation (seven of 56 cases tested). The aim of this paper was to demonstrate that auto-Abs anti-G-CSF revealed in the blood of newborns were produced during foetal life. MATERIALS AND METHODS: Mononuclear cells from cord blood samples of different newborns containing high titer anti-G-CSF Abs were infected with Epstein-Barr virus in vitro, and EBV-immortalized B-cell lines were isolated and characterized for specific anti-G-CSF Ab production. RESULTS: Six different, unrelated cell lines of male origin which showed the presence of EBNA-2 antigen in the nucleus, displayed a B-cell phenotype (CD30+, CD5-, CD10-, HLA-DR+, CD19+, CD20+, CD23+, CD38+, CD25+), coexpressed low intensity sIgM and sIgD, and produced only IgM with prevailing lambda clonal restriction and anti-rhG-CSF Ab reactivity. The Ab specificity was proven against either glycosylated or unglycosylated G-CSF by saturable binding in direct enzyme-linked immunosorbent assays, by competition binding and Western immunoblotting assays. CONCLUSION: The secreted Abs did not affect the in vitro generation of granulocyte colonies by human normal adult haemopoietic progenitor cells in soft agar clonogenic assays, suggesting that these Abs were not neutralizing.

Antibodies binding granulocyte-macrophage colony stimulating factor produced by cord blood-derived B cell lines immortalized by Epstein-Barr virus in vitro.

We detected natural antibodies (auto-Abs) binding human granulocyte-macrophage colony stimulating factor (GM-CSF) in umbilical cord blood (CB) (23 of 94 samples screened) and peripheral blood of women at the end of pregnancy (6 of 42 samples tested). To demonstrate that Abs detected in CB were produced by the fetus, CB mononuclear cells were infected with Epstein-Barr virus in vitro. Ten cell lines producing constitutively anti-recombinant human GM-CSF (rhGM-CSF) Abs were isolated and characterized. These cells displayed a male karyotype, an early activated B cell phenotype, coexpressed surface IgM and IgD, and secreted only IgM with prevailing lambda clonal restriction. Specific cell surface binding of biotinylated rhGM-CSF and high-level anti-rhGM-CSF IgM Ab production were typical features of early cell cultures. In late cell passages the frequency of more undifferentiated B cells increased. Serum Abs of either maternal or fetal origin or Abs produced in culture did not affect the granulocyte and macrophage colony stimulating activity of rhGM-CSF from bone marrow progenitors in soft agar, suggesting that the Abs produced were nonneutralizing.

Design, synthesis, and conformational studies of the hGM-CSF derived peptide (13-27)-Gly-(75-87).

An analogue of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF), hGM-CSF(13-27)-Gly-(75-87) was synthesized by solid phase methodology. This analogue was designed to comprise helices A and C of the native growth factor, linked by a glycine bridge. Helices A and C form half of a four-helix bundle motif in the crystal structure of the native factor and are involved in the interaction with alpha- and beta-chains of the heterodimeric receptor. A conformational analysis of the synthetic analogue by CD, two-dimensional nmr spectroscopy, and molecular dynamics calculations is reported. The analogue is in a random structure in water and assumes a partially alpha-helical conformation in a 1 : 1 trifluoroethanol/water mixture. The helix content in this medium is approximately 70%. By 2D-nmr spectroscopy, two helical segments were identified in the sequences corresponding to helices A and C. In addition to medium- and short-range NOESY connectivities, a long-range cross peak was found between the Cbeta proton of Val(16) and NH proton of His(87) (using the numbering of the native protein). Experimentally derived interproton distances were used as restraints in molecular dynamics calculations, utilizing the x-ray coordinates as the initial structure. The final structure is characterized by two helical segments in close spatial proximity, connected by a loop region. This structure is similar to that of the corresponding domain in the x-ray structure of the native growth factor in which helices A and C are oriented in an antiparallel fashion. The N-terminal residues Gly-Pro of helix C are involved in an irregular turn connecting the two helical segments. As a consequence, helix C is appreciably shifted and slightly rotated with respect to helix A compared to the x-ray structure of the native growth factor. These small differences in the topology of the two helices could explain the lower biological activity of this analogue with respect to that of the native growth factor.

Human GM-CSF interaction with the alpha-chain of its receptor studied using surface plasmon resonance.

A surface plasmon resonance (SPR) based biosensor has been used for studying the interaction of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) with genetically engineered alpha-chain subunits of its specific receptor (GM-Ralpha). Western blot analysis of GM-Ralpha confirmed the correct size (80 kDa) and reactivity of these proteins against anti-GM-Ralpha polyclonal or monoclonal antibodies. GM-CSF was immobilized, using standard amine coupling methods, to the dextran-modified gold biosensor surface in order to capture GM-Ralpha subsequently injected over the sensing layer. GM-Ralpha were shown to specifically form complexes with the immobilized ligand. Pre-incubation of constant amounts of GM-Ralpha with dilutions of soluble GM-CSF before injection of the mixture over the GM-CSF matrix, prevented ligand binding in a dose dependent manner. In contrast, unrelated soluble cytokines or serum proteins (e.g. G-CSF, albumin, etc.) were found to exert no inhibition. Complexes formation blockage by pre-incubation of constant amounts of GM-Ralpha with dilutions of neutralizing anti-GM-Ralpha antibodies was concentration dependent, further assessing the specificity of the interaction. To investigate the possibility of relating the effect on binding affinity of critical conformational changes at the contact site, experiments of multisite binding were performed, flowing a set of neutralizing monoclonal antibodies reacting to different epitopes on GM-CSF over the GM-CSF matrix, before injecting GM-Ralpha. The results indicated that antibody interaction with helix D and helix A of GM-CSF markedly inhibited GM-CSF binding to GM-Ralpha. Comparable results were obtained using the biosensor technology and enzyme-linked immunoassays, in representative experiments performed with the same reagents. These experiments demonstrate that SPR can be successfully used for studying complementary interactions between GM-CSF and its receptor alpha-chains in solution without using labels or secondary tracers and, compared with conventional immunoanalysis methods, significantly saving time.

Detection of digitalis compounds using a surface plasmon resonance-based biosensor.

An automated surface plasmon resonance-based biosensor system has been used to detect endogenous and exogenous digitalis-like factors (EDLF) in the pmolar range in real time. EDLF was purified from umbilical cord blood. EDLF has been suggested to play a role in hypertension and in perinatal adaptation. Highly specific polyclonal anti-ouabain antibodies showed a high affinity binding capacity for ouabain, ouabagenin and strophantidin with an IC50 value of 5 x 10(-10) M, 7.0 x 10(-10) M and 2 x 10(-8) M, respectively. EDLF cross-reacted with antibodies and its concentration in plasma at IC50 was around 50 pmol ouabain equivalent. This study shows the potential usefulness of the biosensor technology for biomolecular interaction analysis. The features of this technology (fully automated, measured in real time, sharpened response) offer several advantages compared with a traditional immunoassay like radioimmunoassay (RIA) in the detection of digitalis compounds in human fluids.

Structure-activity analysis of C-terminal endothelin analogues.

Several synthetic endothelin (ET) analogues of the C-terminal ET hexapeptide (ET16-21) were analyzed by radio-receptor competition binding assays and biologic activity using both ETA and ETB receptor subtypes. In addition, we produced a hybridoma monoclonal antibody, anti-ET15-21, that appeared to crossreact with the entire ET molecule and was able to neutralize its biologic activity. Antibody binding was measured with competition enzyme-linked immunosorbent assays and a surface plasmon resonance-based biosensor (BIA technology). The ET16-21 moiety was modified with systematic replacement of each residue by alanine (Ala-scan). Whereas the C-terminal residues (Asp18, Ile20, and particularly Trp21) were very important for both receptor binding and immunologic activity, Ala substitution in positions 16, 17, and 19 hardly affected such activities. Analysis of another series of synthetic ET16-21 analogues with the His16 residue replaced by a non-amino-acidic block confirmed that the last two C-terminal residues are essential for receptor and antibody binding, whereas the central region of this hexapeptide is much more tolerant to modification. However, a critical steric conformation of the active hexapeptide is necessary.

Natural and therapeutically-induced antibodies to cytokines.

Serum samples obtained from non-immunocompromised patients treated therapeutically with recombinant cytokines (e.g. Il-1alpha; Il-1beta; Il-2 to Il-18; IFNalpha; GM-CSF; G-CSF; etc.) often reveal the presence of high affinity anti-cytokine antibodies. Antibody Fab binding in a saturable manner by ELISA and RIA or western immunoblotting prove their specificity. Antibody level often increases in these patients with repeated cytokine administration, suggesting boosts of antigen stimulation. However, the appearance in circulation of auto-antibodies to exogenous cytokine is not always associated with a decreased clinical response to therapy. The demonstration that non-neutralizing auto-antibodies to several natural cytokines can be found even in sera of normal healthy individuals never treated before with cytokines and particularly during the last trimester of pregnancy and in cord-blood, suggests that these naturally- occurring and therapeutically-induced auto-antibodies may exert different functions, not only as inhibitors or antagonists but also as beneficial physiological cytokine carriers or regulators of their activity.

Toward the rational development of peptidomimetic analogs of the C-terminal endothelin hexapeptide: development of a theoretical model.

In an early report on the structure-activity relationship of endothelin (ET) peptides, it was reported that the C-terminal hexapeptide ET(16-21), His-Leu-Asp-Ile-Ile-Trp, is the minimum ET fragment which maintains biological activity in some, but not all the tissues responding to ETs. Subsequently, other authors described a series of analogs of this peptide, in which the His 16 residue was replaced by non-natural amino acids, characterized by bulky aromatic side chains. Among them, two well-characterized non-selective ETA/ETB antagonists were PD 142893 and PD 145065; interest in these potent ET antagonists was, however, reduced by their peptidic structure which was likely to lead to undesirable properties such as poor bioavailability and short duration of action. On the basis of these premises, our previous studies led to the development of a peptidomimetic ligand of ET receptors (compound 3), based on the replacement of the His 16 residue of ET(16-21) with an (E)-N-(benzyloxy)iminoacyl moiety; compound 3 proved to possess a certain affinity for ET receptors, albeit lower than that shown by PD 142893 and PD 145065. We report here on ETA/ETB binding affinity of compounds 4-12, designed as a new series of ET(16-21) analogs. Compounds 4 and 5 were practically devoid of any affinity; derivatives 6-12 exhibited appreciable affinity indices for ETB receptors higher than that shown by 3, even if still lower than that obtained for PD 145065. This paper also describes the development of a pharmacophoric model able to explain the ET receptor binding properties of our hexapeptide analogs compared with those of PD 142893 and PD 145065 and IRL2500, recently reported as a potent ETB selective endothelin antagonist.

Design, synthesis and conformational analysis of hGM-CSF(13-31)-Gly-Pro-Gly-(103-116).

On the basis of the X-ray structure and results from structure-activity relationship studies, the following GM-CSF analogue was designed and synthesized by solid-phase methodology: hGM-CSF[13-31]-Gly-Pro-Gly-[103-116]-NH2. This analogue was constructed to comprise helices A and D of the native hGM-CSF, covalently linked in an antiparallel orientation by the tripeptide spacer Gly-Pro-Gly, which is known as a turn-inducing sequence. The conformational analysis of the analogue by CD spectroscopy revealed an essentially random structure in water, while alpha-helix formation was observed upon addition of TFE. In 40% TFE the helix content was approximately 45%. By two-dimensional NMR experiments in 1:1 water/trifluoroethanol mixture two helical sequences were identified comprising the segments corresponding to helix A and helix D. In addition to medium-range NOESY connectivities, a long-range cross-peak was found involving the leucine residues at positions 13 and 35. Based on the experimentally derived data (54 NOEs), the structure was refined by restrained molecular dynamics simulations over 120 ps at various temperatures. A representative conformation derived from the computer simulation is mainly characterized by two helical segments connected by a loop region. The overall three-dimensional structure of the analogue is comparable to the X-ray structure of hGM-CSF in that helices A and D are oriented in an antiparallel fashion, forming a two alpha-helix bundle. Nevertheless, there are small differences in the topology of the helices between the solution structure of the designed analogue and the X-ray structure of hGM-CSF. The possible implications of these conformational features at the effects of biological activity are discussed.

Conformation of four peptides corresponding to the alpha-helical segments of human GM-CSF.

The conformation of segments corresponding to the four alpha-helical stretches found in human granulocyte-macrophage colony-stimulating factor was studied in water solution in the presence of different amounts of 2,2,2-trifluoroethanol (TFE). The CD spectra reveal the onset of secondary structure upon addition of TFE. The final amount of helical conformation varies among the four peptides. In all cases, the conformational transition is complete before 50% TFE (v/v). 1H-NMR studies were conducted at this solvent composition, leading to the assignment of all the resonances and to the definition of the secondary structure for all four fragments.

Naturally occurring and therapy-induced antibodies to human granulocyte colony-stimulating factor (G-CSF) in human serum.

Sera were obtained from two groups of patients. Group A included 7 patients with low-grade non-Hodgkin's lymphoma treated with three or more cycles of standard-dose chemotherapy and recombinant human granulocyte-colony stimulating factor (rhG-CSF). The cytokine was administered to half the patients after the first chemotherapy cycle and to the other half after the second according to a randomized design and then to all patients from the third chemotherapy cycle on, until documented hemopoietic reconstitution. Group B included 3 patients with high-grade non-Hodgkin's lymphoma, 1 patient with resistant Hodgkin's disease, and 1 patient with multiple myeloma who received high-dose chemotherapy and rhG-CSF. Anti-G-CSF antibodies were detected in the sera of 4 patients. Both immunoglobulin IgM and IgG antibodies were detected at low levels in pretreatment sera from one group A patient. IgG antibody titers increased markedly during the first and second periods of G-CSF administration. IgG class antibodies developed in 3 groups B patients during the first course of rhG-CSF administration. Circulating anti-G-CSF antibodies did not seem to affect hematological recovery. Low levels of anti-G-CSF antibodies were also detected in sera (15/135) from different healthy adults and in sera (5/40) from umbilical cord blood. Saturable antibody binding and competition enzyme-linked immunosorbent assay (ELISA) and immunoblotting confirmed antibody specificity.

Detection and epitope mapping of immunoreactive human endothelin-1 using ELISA and a surface plasmon resonance-based biosensor.

A surface plasmon resonance-based biosensor (BIA technology) and enzyme-linked immunosorbent assays (ELISA) have been used for detecting and characterizing human endothelin (ET), a potent vasoactive 21 amino acid polypeptide. Antibodies produced against the isoform ET-1 and its C-terminal eptapeptide ET-1(15-21) have been characterized with respect to their binding capacity to the two isoforms ET-1 and ET-3, the non-secreted portion of the precursor molecule Big.ET-1(22-38), the C-terminal of ET-1, six analogues of ET-1(16-21) each containing a substitution with Ala of a single amino acid in positions 16-21, respectively, and three synthetic cyclic peptides mimicking the N-terminal portion of ET-1. Antibodies reacting with ET-1 also bound to ET-1(16-21) and, with less affinity, to ET-3 but did not cross-react with Big.ET-1(22-38). Ala substitution in positions 16, 17 and 19 of ET-1(16-21) hardly affected the antibody binding capacity of ET-1(16-21), whereas Ala substitution of Asp18, Ile20 and, in particular, Trp21, inhibited its immunoreactivity. The C-terminus thus represents an immunodominant epitope in ET-1 and is important for antibody binding. Epitope mapping using as antibody pairs polyclonal anti-ET-1 and monoclonal anti-ET-1(15-21) antibodies indicated the presence of another immunogenic domain in the N-terminal portion of the molecule. There was excellent agreement between the epitopes determined using ELISA and BIA analyses.

Natural and therapy-induced anti-GM-CSF and anti-G-CSF antibodies in human serum.

Serum samples were obtained from patients with lymphoid and plasma cell malignancies who received after chemotherapy human recombinant GM-CSF or G-CSF. Sera from some patients revealed the presence of anti-cytokine antibodies, particularly after repetitive cytokine injections. Antibody Fab binding in a saturable manner by ELISA and Western immuno-blotting confirmed antibody specificity. Anti-cytokine antibodies were detected before the exogenous cytokine injections in some patients, but increasing antibody levels were found after one or subsequent treatments. Low levels of anti-GM-CSF and anti-G-CSF antibodies were also detected in a relatively large proportion (about 10-30%) of normal sera from different adult healthy volunteers who had never been treated before with exologous cytokines as well as from cord blood. EBV-immortalized cord blood derived B-cell cultures were also found to produce anti GM-CSF and/or anti-G-CSF antibodies with high frequency.

Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct epitopes to be identified over ELISA.