Characterization of the interaction between Robo1 and heparin and other glycosaminoglycans

Roundabout 1 (Robo1) is the cognate receptor for secreted axon guidance molecule, Slits, which function to direct cellular migration during neuronal development and angiogenesis. The Slit2–Robo1 signaling is modulated by heparan sulfate, a sulfated linear polysaccharide that is abundantly expressed on the cell surface and in the extracellular matrix. Biochemical studies have further shown that heparan sulfate binds to both Slit2 and Robo1 facilitating the ligand–receptor interaction. The structural requirements for heparan sulfate interaction with Robo1 remain unknown. In this report, surface plasmon resonance (SPR) spectroscopy was used to examine the interaction between Robo1 and heparin and other GAGs and determined that heparin binds to Robo1 with an affinity of ∼650 nM. SPR solution competition studies with chemically modified heparins further determined that although all sulfo groups on heparin are important for the Robo1–heparin interaction, the N-sulfo and 6-O-sulfo groups are essential for the Robo1–heparin binding. Examination of differently sized heparin oligosaccharides and different GAGs also demonstrated that Robo1 prefers to bind full-length heparin chains and that GAGs with higher sulfation levels show increased Robo1 binding affinities.     

Avidin is a heparin-binding protein. Affinity,specificity and structural analysis

The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglycans (GAGs) have been investigated using heparin-coated microtiter-plate assays, a filter binding assay and surface plasmon resonance (SPR) analysis using a BIAcore 2000 biosensor. Avidin binds heparin and heparan sulfate, and chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate or hyaluronan were unable to compete for binding. Highest-affinity binding was observed with heparin, and weaker binding was seen when using heparan sulfate or low molecular weight heparin preparations. This indicated that only specific polysaccharide structures tightly interact with avidin. Approximately two avidin molecules bind to each heparin molecule with an overall affinity of 160 nM. The interaction is pH dependent, increasing five-fold upon decreasing the pH from 7.5 to 5.5, while binding was negligible at pH 9. We demonstrate the potential of fluorescent avidin derivatives as a tool for the detection of heparin and heparan sulfates on surfaces by application to both heparin immobilized on polystyrene plates and heparan sulfate on cell surfaces.     

A dot blot assay for sulfated glycosaminoglycans

A dot blot assay for detection of low amounts of heparin and sulfated glycosaminoglycans (GAGs) is described. The detection range is between 25 ng/ml and 1000 ng/ml of heparin. The assay is based on the interference of sulfated GAGs with the binding of a synthetic ligand (described in this paper) to defined receptors like collagen type V and histones. Ligand binding to type V collagen was suppressed specifically by heparin, but not by other sulfated GAGs like heparin sulfate and chondroitin sulfate. Ligand binding to histones was suppressed most strongly by heparin, but also by chondroitin sulfate. Hyaluronic acid did not interfere.     

Sugar Chips immobilized with synthetic sulfated disaccharides of heparin/heparan sulfate partial structure

Carbohydrate chip technology has a great potential for the high-throughput evaluation of carbohydrate-protein interactions. Herein, we report syntheses of novel sulfated oligosaccharides possessing heparin and heparan sulfate partial disaccharide structures, their immobilization on gold-coated chips to prepare array-type Sugar Chips, and evaluation of binding potencies of proteins by surface plasmon resonance (SPR) imaging technology. Sulfated oligosaccharides were efficiently synthesized from glucosamine and uronic acid moieties. Synthesized sulfated oligosaccharides were then easily immobilized on gold-coated chips using previously reported methods. The effectiveness of this analytical method was confirmed in binding experiments between the chips and heparin binding proteins, fibronectin and recombinant human von Willebrand factor A1 domain (rh-vWf-A1), where specific partial structures of heparin or heparan sulfate responsible for binding were identified.     

Biophysical characterization of glycosaminoglycan-IL-7 interactions using SPR

Glycosaminoglycans (GAGs) interact with a number of cytokines and growth factors thereby playing an essential role in the regulation of many physiological processes. These interactions are important for both normal signal transduction and the regulation of the tissue distribution of cytokines/growth factors. In the present study, we employed surface plasmon resonance (SPR) spectroscopy to dissect the binding interactions between GAGs and murine and human forms of interleukin-7 (IL-7). SPR results revealed that heparin binds with higher affinity to human IL-7 than murine IL-7 through a different kinetic mechanism. The optimal oligosaccharide length of heparin for the interactions to human and murine IL-7 involves a sequence larger than a tetrasaccharide. These results further demonstrate that while IL-7 is principally a heparin/heparan sulfate binding protein, it also interacts with dermatan sulfate, chondroitin sulfates C, D, and E, indicating that this cytokine preferentially interacts with GAGs having a higher degree of sulfation.     

Glycosaminoglycans from bovine eye vitreous humour and interaction with collagen type II

Glycosaminoglycans (GAGs) play an important role in stabilizing the gel state of eye vitreous humour. In this study, the composition of GAGs present in bovine eye vitreous was characterized through disaccharide analysis by liquid chromatography-mass spectrometry. The interaction of GAGs with collagen type II was assessed using surface plasmon resonance (SPR). The percentage of hyaluronic acid (HA), chondroitin sulfate (CS) and heparan sulfate (HS), of total GAG, were 96.2%, 3.5% and 0.3%, respectively. The disaccharide composition of CS consisted of 4S (49%), 0S (38%) 6S (12%), 2S6S (1.5%) and 2S4S (0.3%). The disaccharide composition of HS consisted of 0S (80%), NS2S (7%), NS (7%), 6S (4%), NS6S (2%), and TriS, 2S and 4S6S (each at 0.1%). The average molecular weights of CS and HS were 148 kDa and 204 kDa, respectively. SPR reveals that collagen type II binds to heparin (primarily composed of TriS) with a binding affinity (K _D) of 755 nM and interacts with other GAGs, including CSB and CSE. Both bovine vitreous CS and HS interact with collagen type II, with vitreous HS showing a higher binding affinity.     

Binding of interferon gamma by glycosaminoglycans: a strategy for localization and/or inhibition of its activity.

Glycosaminoglycans (GAGs) are a group of negatively charged molecules present in many tissues as components of the extracellular matrix, basement and cellular membranes. This work analysed the ability of this group of substances to interact with human interferon gamma and the effect of those interactions on its biologic activity. A variety of GAGs (heparin, heparan sulfate, chondroitin sulfate and hyaluronic acid), and a related sulfated polysaccharide (dextran sulfate), were found to interact with IFN-gamma as determined by inhibition of the binding of [125I]IFN-gamma to COLO-205 cells and binding to wells coated with GAGs. These interactions were inhibited by synthetic peptides mimicking the sequences of the basic amino acid cluster located at the C-terminal end of mouse and human IFN-gamma, or by poly-L-lysine, suggesting that ionic interactions between the positively-charged C-terminus and negatively charged groups in GAGs were involved. IFN-gamma molecules bound to plate-immobilized or endothelial cell surface GAGs retained biological activity, since they could induce major histocompatibility complex (MHC) class II expression on COLO-205 cells, suggesting that cell surface GAGs might be able to present IFN-gamma to its receptors. These results suggest important regulatory roles for GAGs on the activity of IFN-gamma in vivo.     

LTBP-2 has multiple heparin/heparan sulfate binding sites

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of poorly understood function associated with fibrillin-1-containing microfibrils during elastinogenesis. In this study we investigated the molecular interactions of LTBP-2 with heparin and heparan sulfate proteoglycans (HSPGs) since unidentified cell surface HSPGs are critical for normal fiber assembly. In solid phase assays, heparin conjugated to albumin (HAC) bound strongly to recombinant full-length human LTBP-2. This interaction was completely blocked by addition of excess heparin, but not chondroitin sulfate, confirming specificity. Analysis of binding to LTBP-2 fragments showed that HAC bound strongly to N-terminal fragment LTBP-2 NT(H) and more weakly to central fragment LTBP-2 C(H). No binding was detected to C-terminal fragment LTBP-2 CT(H). Kds for heparin binding were calculated for full-length LTBP-2, LTBP-2 NT(H) and LTBP-2 C(H) as 0.9 nM, 0.7 nM and 80 nM respectively. HAC interaction with fragment LTBP-2 NT(H) was not sensitive to EDTA or EGTA indicating that binding had no requirement for Ca²⁺ ions whereas HAC binding to fragment LTBP-2 C(H) was markedly reduced by these chelating agents indicating a degree of Ca²⁺ dependence. Inhibition studies with synthetic peptides identified three major heparin binding sequences in fragment LTBP-2 NT(H), including sequence LTEKIKKIKIV in the first large cysteine-free domain of LTBP-2, adjacent to the previously identified fibulin-5 binding site. LTBP-2 was found to interact strongly in a heparin-inhibitable manner with cell surface HSPG syndecan-4, but showed no interaction with recombinant syndecan-2. LTBP-2 also showed strong interaction with the heparan sulfate chains of basement membrane HSPG, perlecan. The potential importance of HSPG–LTBP-2 interactions in elastic fiber assembly and microfibril attachment to basement membranes is discussed.     

Characterization of osteoprotegerin binding to glycosaminoglycans by surface plasmon resonance: role in the interactions with receptor activator of nuclear factor kappaB ligand (RANKL) and RANK

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor kappaB ligand (RANKL), a key inducer of osteoclastogenesis via its receptor RANK. We previously showed that RANK, RANKL, and OPG are able to form a tertiary complex and that OPG must be also considered as a direct effector of osteoclast functions. As OPG contains a heparin-binding domain, the present study investigated the interactions between OPG and glycosaminoglycans (GAGs) by surface plasmon resonance and their involvement in the OPG functions. Kinetic data demonstrated that OPG binds to heparin with a high-affinity (KD: 0.28 nM) and that the pre-incubation of OPG with heparin inhibits in a dose-dependent manner the OPG binding to the complex RANK-RANKL. GAGs from different structure/origin (heparan sulfate, dermatan sulfate, and chondroitin sulfate) exert similar activity on OPG binding. The contribution of the sulfation pattern and the size of the oligosaccharide were determined in this inhibitory mechanism. The results demonstrated that sulfation is essential in the OPG-blocking function of GAGs since a totally desulfated heparin loses its capacity to bind and to block OPG binding to RANKL. Moreover, a decasaccharide is the minimal structure that totally inhibits the OPG binding to the complex RANK-RANKL. Western blot analysis performed in 293 cells surexpressing RANKL revealed that the pre-incubation of OPG with these GAGs strongly inhibits the OPG-induced decrease of membrane RANKL half-life. These data support an essential function of the related glycosaminoglycans heparin and heparan sulfate in the activity of the triad RANK-RANKL-OPG.     

Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

ABSTRACT: BACKGROUND: Many growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans (GAGs), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. RESULTS: Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse[trade mark sign]). CONCLUSIONS: A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2 osteogenic activity for in vivo de novo bone generation when delivered together on a scaffold as a single-phase. The use of HS/CS PGs may be useful to augment GF therapeutics, and a plasmid-based approach has been shown here to be highly effective.     

Protein kinase CK2: evidence for a protein kinase CK2beta subunit fraction, devoid of the catalytic CK2alpha subunit, in mouse brain and testicles

Apolipoprotein E (apoE), a key lipid transport protein, displays a heparin-binding property that is critical in several apoE functions. The kinetics of the interaction between apoE isoforms and glycosaminoglycans (GAGs) were studied using surface plasmon resonance. The dissociation constant of equilibrium K(D) for apoE3-heparin interaction was estimated to be 12 nM for apoE3 and three common apoE isoforms revealed similar affinities for heparin. ApoE binds to GAGs in the following order: heparin>heparan sulfate>dermatan sulfate>chondroitin sulfate. The affinity parameter of the binding of low molecular weight heparins to apoE is correlated with the chain length. The effective number Z of electrostatic interactions between plasma apoE3 and heparin was assessed to be three. Metal chelators were able to diminish apoE-binding to heparin, suggesting some stabilizing effect of metal ions while reconstitution with lipids did not affect binding affinities for heparin, suggesting that the N-terminal heparin-binding site is responsible for apoE-containing lipoprotein interactions with heparin.     

Specific inhibition of binding of antistasin and [A103,106,108] antistasin 93-119 to sulfatide (Gal(3-SO4)beta 1-1Cer) by glycosaminoglycans.

Leech-derived antistasin is a potent anticoagulant and antimetastatic protein that binds sulfatide (Gal(3-SO4)beta 1-1Cer) and sulfated polysaccharides. In this study, the synthetic fragment [A103,106,108] antistasin 93-119, which corresponds to the carboxyl terminus, showed specific and saturable binding to sulfatide. Binding was competitively blocked by glycosaminoglycans (GAGs) in the order: dextran sulfate 5000 congruent to dextran sulfate 500,000 greater than heparin greater than dermatan sulfate much greater than chondroitin sulfates A and C. This rank order of inhibitory potency was identical to that observed with whole antistasin. We suggest that residues 93-119 of antistasin represent a critical domain for binding GAGs and sulfated glycolipids.     

Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases

Sulfated glycans play critical roles during the development, differentiation and growth of various organisms. The most well-studied sulfated molecules are sulfated glycosaminoglycans (GAGs). Recent incidents of heparin drug contamination convey the importance of having a convenient and sensitive method for detecting different GAGs. Here, we describe a molecular method to detect GAGs in biological and biomedical samples. Because the sulfation of GAGs is generally not saturated in vivo, it is possible to introduce the radioisotope (35)S in vitro using recombinant sulfotransferases, thereby allowing detection of minute quantities of these molecules. This strategy was also successfully applied in the detection of other glycans. As examples, we detected contaminant GAGs in commercial heparin, heparan sulfate and chondroitin samples. The identities of the contaminant GAGs were further confirmed by lyase digestion. Oversulfated chondroitin sulfate was detectable only following a simple desulfation step. Additionally, in vitro sulfation by sulfotransferases allowed us to map glycan epitopes in biological samples. This was illustrated using mouse embryo and rat organ tissue sections labeled with the following carbohydrate sulfotransferases: CHST3, CHST15, HS3ST1, CHST4 and CHST10.     

Kinetic and Structural Studies on the Interactions of Heparin and Proteins of Human Seminal Plasma using Surface Plasmon Resonance

Heparin is naturally occurring polysaccharides which interacts with seminal plasma proteins and regulate multiple steps in fertilization process. Qualitative and quantitative information regarding the affinity for heparin-seminal plasma proteins interactions is not generally well documented and there are no reports of a comprehensive analysis of these interactions in human seminal plasma. Such information should improve our understanding of how GAGs especially heparin present in the reproductive tract regulate fertilization. In this study, we use SPR to study interactions of heparin with various seminal plasma heparin-binding proteins (HBPs). HBPs like lactoferrin (LF), fibronectin fragment (FNIII), semenogelinI (SGI) and prostate specific antigen (PSA) all bind heparin with different binding kinetics and affinities. Kinetic data suggests that FNIII binds heparin with a high affinity (KD=3.2 nM), while PSA binds heparin with a micromolar affinity (KD=11.1 μM). Preincubation of SGI with heparin inhibits the binding of SGI to immobilized PSA in a dosedependent manner, while FNIII incubated with heparin binds with an increased affinity to PSA. Solution-competition studies show that the minimum size of a heparin oligosaccharide capable of binding with PSA is greater than a tetrasaccharide, with LF and SGI is larger than a hexasaccharide and for FNIII is larger than an octasaccharide.     

Glycosaminoglycans from chicken muscular stomach or gizzard

Glycosaminoglycans (GAGs) were prepared from the muscular stomach or gizzard of the chicken. The content of GAGs on a dry weight basis contains 0.4 wt.% a typical value observed for a muscle tissue. The major GAG components were chondroitin-6-sulfate and chondroitin-4-sulfate (~64 %) of molecular weight 21–22 kDa. Hyaluronan (~24 %) had a molecular weight 120 kDa. Smaller amounts (12 %) of heparan sulfate was also present which was made of more highly sulfated chains of molecular weight of 21-22 kDa and a less sulfated low molecular weight (< 10 kDa) heterogeneous partially degraded heparan sulfate. Chicken gizzard represents an inexpensive and readily available source of muscle tissue-derived GAGs.     

Interaction of 70-kDa heat shock protein with glycosaminoglycans and acidic glycopolymers

Interaction of Hsp70 with natural and artificial acidic glycans is demonstrated based on the native PAGE analysis. Hsp70 interacts with acidic glycopolymers that contain clustered sulfated and di-sialylated glycan moieties on a polyacrylamide backbone, but not with neutral or mono-sialylated glycopolymers. Hsp70 also interacts and forms a large complex with heparin, heparan sulfate, and dermatan sulfate that commonly contain 2-O-sulfated iduronic acid residues, but not with other types of glycosaminoglycans (GAGs). Hsp70 consists of the N-terminal ATPase domain and the C-terminal peptide-binding domain. The interaction analyses using the recombinant N- and C-terminal half domains show that the ATPase domain mediates the direct interaction with acidic glycans, while the peptide-binding domain stabilizes the large complexes with particular GAGs. To our knowledge, this is the first demonstration of direct binding of Hsp70 to the particular GAGs. This property may be involved in the physiological functions of Hsp70 at the plasma membrane and extracellular environments.     

Trypanosoma cruzi heparin-binding proteins mediate the adherence of epimastigotes to the midgut epithelial cells of Rhodnius prolixus

Heparin-binding proteins (HBPs) have been demonstrated in both infective forms of Trypanosoma cruzi and are involved in the recognition and invasion of mammalian cells. In this study, we evaluated the potential biological function of these proteins during the parasite-vector interaction. HBPs, with molecular masses of 65·8 kDa and 59 kDa, were isolated from epimastigotes by heparin affinity chromatography and identified by biotin-conjugated sulfated glycosaminoglycans (GAGs). Surface plasmon resonance biosensor analysis demonstrated stable receptor-ligand binding based on the association and dissociation values. Pre-incubation of epimastigotes with GAGs led to an inhibition of parasite binding to immobilized heparin. Competition assays were performed to evaluate the role of the HBP-GAG interaction in the recognition and adhesion of epimastigotes to midgut epithelial cells of Rhodnius prolixus. Epithelial cells pre-incubated with HBPs yielded a 3·8-fold inhibition in the adhesion of epimastigotes. The pre-treatment of epimastigotes with heparin, heparan sulfate and chondroitin sulfate significantly inhibited parasite adhesion to midgut epithelial cells, which was confirmed by scanning electron microscopy. We provide evidence that heparin-binding proteins are found on the surface of T. cruzi epimastigotes and demonstrate their key role in the recognition of sulfated GAGs on the surface of midgut epithelial cells of the insect vector.     

Modulation of blood coagulation and fibrinolysis by polyamines in the presence of glycosaminoglycans

The effects of polyamines on blood coagulation and fibrinolysis in the presence of glycosaminoglycans (GAGs) were examined because it is known that heparin (HP) interacts with polyamines, especially with spermine. Spermine was able to reverse the prolongation of coagulation time of rabbit plasma caused by HP. The effects of various GAGs on thrombin activity in the presence of anti-thrombin III (AT) were then tested using a synthetic substrate. Inhibition of thrombin activity by GAGs was in the order HP > heparan sulfate (HS) > dermatan sulfate (DS) > chondroitin sulfate (CS) approximately hyaluronan (HA). When these GAGs were fully sulfonated, the inhibitory activity of HS, DS, CS and HA, but not HP, became stronger. The effects of GAGs on thrombin activity were reversed by polyamines, in particular spermine. The EC(50) value of spermine for reversal of HP inhibition was 30-50 microM, and the K(d) value of spermine for heparin was 41.1 microM. Analysis by surface plasmon resonance (SPR) indicated that the interaction between AT and HP was weakened by spermine through its binding to HP. The effect of HP on fibrinolysis was then examined. When Glu-plasminogen and tissue-type plasminogen activator (tPA) were used as enzyme source, HP strongly enhanced the plasmin activity and spermine reversed this effect. Analysis by SPR suggests that the structure of the active site of tPA may be changed through the ternary complex formation of tPA, HP and spermine. The results indicate that blood coagulation was enhanced and fibrinolysis was weakened by spermine in the presence of HP.     

Structural determinants of heparan sulfate interactions with Slit proteins

We have previously demonstrated that the Slit proteins, which are involved in axonal guidance and related processes, are high-affinity ligands of the heparan sulfate proteoglycan glypican-1. Glypican-Slit protein interactions have now been characterized in greater detail using two approaches. The ability of heparin oligosaccharides of defined structure (ranging in size from disaccharide to tetradeccasaccharide) to inhibit binding of a glypican-Fc fusion protein to recombinant human Slit-2 was determined using an ELISA. Surface plasmon resonance (SPR) spectroscopy, which measures the interactions in real time, was applied for quantitative modeling of heparin-Slit binding on heparin biochips. Heparin was covalently immobilized on these chips through a pre-formed albumin-heparin conjugate, and the inhibition of Slit binding by heparin, LMW heparin, and heparin-derived oligosaccharides (di-, tetra-, hexa-, and octa-) was examined utilizing solution competition SPR. These competition studies demonstrate that the smallest heparin oligosaccharide competing with heparin binding to Slit was a tetrasaccharide, and that in the ELISA maximum inhibition (approximately 60% at 2 microM concentration) was attained with a dodecasaccharide.     

Dextran sulfate and heparin interact with CD4 molecules to inhibit the binding of coat protein (gp120) of HIV

Dextran sulfate, heparin, and certain other sulfated polysaccharides potently inhibit the adsorption of HIV to CD4+ cells. The mechanism of this inhibition is unclear and, specifically, it is unknown if these agents act at the level of CD4-gp120 binding. For example, previous reports have demonstrated that dextran sulfate does not inhibit the cell surface binding of anti-CD4 mAb known to be directed at the gp120 binding site. In order to confirm and extend these observations, in the present study, it was shown that dextran sulfate does not inhibit the binding of OKT4A, OKT4C, Leu3a, or B66.6 to CD4+ cells as measured by cytofluorography. Next, recombinant forms of CD4 (rT4) and gp120 (rgp120) were utilized to directly study their molecular interaction in the absence of other viral or cellular structures. Reciprocal solid phase ELISA assays were developed to study directly the effects of sulfated polysaccharides on the binding of rT4 to immobilized rgp120 and vice versa. Dextran sulfate, heparin, and fucoidan, but not chondroitin sulfate, inhibited the binding of rgp120 to rT4. Importantly, dextran sulfate and heparin pre-treatment of immobilized rT4, but not immobilized rgp120, inhibited rT4-rgp120 binding. Taken together, these data suggest that while both sulfated polysaccharides and anti-CD4 mAb inhibit gp120 binding, the sulfated polysaccharides interact with sites on CD4 that are distinct from those with which the antibodies bind.