Insulin receptor‐insulin interaction kinetics using multiplex surface plasmon resonance

Type 2 diabetes affects millions of people worldwide, and measuring the kinetics of insulin receptor‐insulin interactions is critical to improving our understanding of this disease. In this paper, we describe, for the first time, a rapid, real‐time, multiplex surface plasmon resonance (SPR) assay for studying the interaction between insulin and the insulin receptor ectodomain, isoform A (eIR‐A). We used a scaffold approach in which anti‐insulin receptor monoclonal antibody 83–7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR‐A capture. The multiplex SPR system (ProteOn XPR36?, Bio‐Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. Interactions between recombinant human insulin followed a two‐site binding pattern, consistent with the literature, with a high‐affinity site (dissociation constant K_D1 = 38.1 ± 0.9 nM) and a low‐affinity site (K_D2 = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants K_D1 = 73.2 ± 1.8 nM and K_D2 = 148.9 ± 6.1 nM, but the fit to kinetic data was improved when we included a conformational change factor in which the high‐affinity site was converted to the low‐affinity site. The new SPR assay enables insulin‐eIR‐A interactions to be followed in real time and could potentially be extended to study the effects of humoral factors on the interaction, without the need for insulin labeling. Copyright © 2013 John Wiley & Sons, Ltd.     

A fusion protein expression analysis using surface plasmon resonance imaging

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the affinity-tagged recombinant proteins expressed in Escherichia coli. With regards to model proteins, the hexahistidine-ubiquitin-tagged human growth hormone (His(6)-Ub-hGH), glutathione S-transferase-tagged human interleukin-6 (GST-hIL6), and maltose-binding protein-tagged human interleukin-6 (MBP-hIL6) expressed in E. coli were analyzed. The cell lysates were spotted on gold thin films coated with 11-mercaptoundecanol (MUOH)/dextran derivatized with Ni(II)-iminodiacetic acid (IDA-Ni(II)), glutathione, or cyclodextrin. After a brief washing of the gold chip, SPR imaging measurements were carried out in order to detect the bound affinity-tagged fusion proteins. Using this new approach, rapid high-throughput expression analysis of the affinity-tagged proteins were obtained. The SPR imaging protein chip system used to measure the expression of affinity-tagged proteins in a high-throughput manner is expected to be an attractive alternative to traditional laborious and time-consuming methods, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots.     

Analysis of the interaction between the aspartic peptidase inhibitor SQAPI and aspartic peptidases using surface plasmon resonance

Aspartic peptidase inhibitors, which are themselves proteins, are strong inhibitors (small inhibition constants) of some aspartic peptidases but not others. However, there have been no studies of the kinetics of the interaction between a proteinaceous aspartic peptidase inhibitor and aspartic peptidases. This paper describes an analysis of rate constants for the interaction between recombinant squash aspartic peptidase inhibitor (rSQAPI) and a panel of aspartic peptidases that have a range of inhibition constants for SQAPI. Purified rSQAPI completely inhibits pepsin at a 1:1 molar ratio of pepsin to rSQAPI monomer (inhibition constant 1 nM). The interaction of pepsin with immobilized rSQAPI, at pH values between 3.0 and 6.0, was monitored using surface plasmon resonance. Binding of pepsin to rSQAPI was slow (association rate constants ca 10(4)M (-1)s(-1)), but rSQAPI was an effective pepsin inhibitor because dissociation of the rSQAPI-pepsin complex was much slower (dissociation rate constants ca 10(-4)s(-1)), especially at low pH values. Similar results were obtained with a His-tagged rSQAPI. Strong inhibition (inhibition constant 3 nM) of one isoform (rSap4) of the family of Candida albicans-secreted aspartic peptidases was, as with pepsin, characterized by slow binding of rSap4 and slower dissociation of the rSap4-inhibitor complex. In contrast, weaker inhibition of the Glomerella cingulata-secreted aspartic peptidase (inhibition constant 7 nM) and the C. albicans rSap1 and Sap2 isoenzymes (inhibition constants 25 and 400 nM, respectively) was, in each case, characterized by a larger dissociation rate constant.     

Surface plasmon resonance spectroscopy in the study of membrane-mediated cell signalling.

Peptide-membrane interactions contribute to many important biological processes such as cellular signaling, protein trafficking and ion-channel formation. During receptor-mediated signalling, activated intracellular signalling molecules are often recruited into receptor-induced signaling complexes at the cytoplasmic surface of the cell membrane. Such recruitment can depend upon protein-protein and protein-lipid interactions as well as protein acylation. A wide variety of biophysical techniques have been combined with the use of model membrane systems to study these interactions and have provided important information on the relationship between the structure of these proteins involved in cell signalling and their biological function. More recently, surface plasmon resonance (SPR) spectroscopy has also been applied to the study of biomembrane-based systems using both planar mono- or bilayers or liposomes. This article provides an overview of these recent applications, which demonstrate the potential of SPR to enhance our molecular understanding of membrane-mediated cellular signalling.     

The investigation of recognition interaction between phenylboronate monolayer and glycated hemoglobin using surface plasmon resonance

Glycated hemoglobin (HbA1c) is formed by a nonenzymatic reaction of glucose with the N-terminal valine of adult hemoglobin's beta-chain. The amount of HbA1c reflects the average concentration of glucose variation level over the preceding 2 to 3 months. Because the boronate has antibody mimicking for HbA1c, often it is used to detect HbA1c. However, factors such as the ratio of the phenylboronic acid derivatives and diol composition, the pH of the solution, and the stereostructure of phenylboronic acid derivatives could influence the interactions between phenylboronic acid derivatives and diol composition. In this study, the factors were evaluated using surface plasmon resonance (SPR). The results show that pH value is an important factor affecting HbA1c and phenylboronic acid to form the complex and Lewis bases. This could change the stereostructure of phenylboronic acid to form B(OH)(3) for binding with saccharine easily. In addition, linear response appeared in HbA1c in the range of 0.43 to 3.49 mug/ml, and the detection limit was 0.01 microg/ml. The results also demonstrated that an SPR biosensor can be used as a sensitive technique for improving the accuracy and correctness of HbA1c measurement.     

A surface plasmon resonance study of the interactions between the component subunits of protein kinase CK2 and two protein substrates,casein and calmodulin

Surface plasmon resonance has been used to study the interaction between the subunits composing protein kinase CK2 (two catalytic, -subunits, and two regulatory, -subunits), as well as the interaction of each subunit with two types of protein substrates, casein, the phosphorylation of which is activated by the regulatory subunit, and calmodulin, which belongs to the kind of substrates on which the catalytic subunit is down regulated by the regulatory subunit. The interaction of casein with the catalytic subunit differs from the interaction with the holoenzyme. Similarly to the interaction with the regulatory subunit, the catalytic subunit interacts with the protein substrate forming a very stable, irreversible complex. The reconstituted holoenzyme, however, binds casein reversibly, displaying a binding mode similar to that displayed by the regulatory subunit. The interaction of calmodulin with the catalytic subunit gives place, like in the case of casein, to an irreversible complex. The interactions with the regulatory subunit, and with the holoenzyme were practically negligible, and the interaction with the regulatory subunit disappeared upon increasing the temperature value to close to 30°C. The presence of polylysine induced a high increase in the extent of calmodulin binding to the holoenzyme. The results obtained suggest that CK2 subunit and protein substrates share a common, or at least an overlapping site of interaction on the catalytic subunit. The interaction between both subunits would prevent substrates from binding irreversibly to subunit, and, at the same time, it would generate a new and milder site of interaction between the whole holoenzyme and the protein substrate. The main difference between casein and calmodulin would consist in the lower affinity display by the last for the new site generated upon the binding of the regulatory subunit, in the absence of polycations like polylysine.     

Evaluation of surface hydrophobicity of immobilized protein with a surface plasmon resonance sensor

Immobilization is widely used to isolate agglutinative and associative proteins with large hydrophobic surfaces. Surface hydrophobicities of immobilized proteins were quantified by measuring the adsorption amounts of Triton X-100 as a hydrophobic probe with a biosensor that utilizes the phenomena of surface plasmon resonance (SPR). We measured SPR signal changes derived from adsorption of Triton X-100 to five kinds proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller equation, partly modified with a term for correcting an influence of the net charge of immobilized protein. SPR signal changes obtained by this method correlated with the values of surface hydrophobicities obtained by conventional assay using a hydrophobic probe. Thus this measuring method using an SPR sensor and Triton X-100 is expected to be a tool for quantifying surface hydrophobicities of immobilized proteins.     

A method of reversible biomolecular immobilization for the surface plasmon resonance quantitative analysis of interacting biological macromolecules

This article presents a new procedure for the immobilization of macromolecules on gold surfaces, with the purpose of studying macromolecular interactions by simple optical configurations rendering surface plasmon resonance. Gold surfaces were covered by a three-layer structure composed of poly-L-lysine irreversibly bound to gold, followed by a second layer of heparin and a third layer of polylysine. The three-layer structure of polylysine-heparin-polylysine remains irreversibly bound to gold, it prevents biomolecules from coming into direct contact with the metal surface, and it allows the irreversible binding of different proteins and polynucleotides. After binding of a macromolecule to the three-layer structure, the interaction with a second macromolecule can be studied, and then the complex formed by the two interacting macromolecules, together with the second heparin layer and the third polylysine layer, can be broken down just by treatment with an alkaline solution having a pH value above the pK value of the amino groups of polylysine. The first polylysine layer remains irreversibly bound to gold, ready to form a new three-layer structure and, therefore, to support a new macromolecular interaction on the same regenerated surface. Polynucleotide interactions, the proteolytic action of chymotrypsin, and the interaction between the component subunits of a heterotetrameric enzyme are described as examples of macromolecular interactions studied by using this system. The method may be especially suitable for developing of low-cost systems aimed to look for surface resonance signals, and it offers the advantage of allowing calculation of parameters related to the size and stoichiometry of the interacting macromolecules, in addition to the kinetic and equilibrium properties of the interaction.     

Monitoring bioactive and total antibody concentrations for continuous process control by surface plasmon resonance spectroscopy

Monoclonal antibodies have become an increasingly important part of fundamental research and medical applications. To meet the high market demand for monoclonal antibodies in the biopharmaceutical sector, industrial manufacturing needs to be achieved by large scale, highly productive and consistent production processes. These are subject to international guidelines and have to be monitored intensely due to high safety standards for medical applications. Surface plasmon resonance spectroscopy — a fast, real‐time, and label‐free bio‐sensing method — represents an interesting alternative to the quantification of monoclonal antibody concentrations by enzyme‐linked immunosorbent assay during monoclonal antibody production. For the application of monitoring bioactive and total monoclonal antibody concentrations in cell culture samples, a surface plasmon resonance assay using a target‐monoclonal antibody model system was developed. In order to ensure the subsequent detection of bioactive monoclonal antibody concentrations, suitable immobilization strategies of the target were identified. A significant decrease of the limit of detection was achieved by using an adapted affinity method compared to the commonly used amine coupling. Furthermore, the system showed limit of detection in the low ng/mL range similar to control quantifications by enzyme‐linked immunosorbent assay. Moreover, the comparison of total to bioactive monoclonal antibody concentrations allows analysis of antibody production efficiency. The development of an alternative quantification system to monitor monoclonal antibody production was accomplished using surface plasmon resonance with the advantage of low analyte volume, shorter assay time, and biosensor reusability by target‐layer regeneration. The established method provides the basis for the technical development of a surface plasmon resonance‐based system for continuous process monitoring.     

Differential binding studies applying functional protein microarrays and surface plasmon resonance

A variety of different in vivo and in vitro technologies provide comprehensive insights in protein-protein interaction networks. Here we demonstrate a novel approach to analyze, verify and quantify putative interactions between two members of the S100 protein family and 80 recombinant proteins derived from a proteome-wide protein expression library. Surface plasmon resonance (SPR) using Biacore technology and functional protein microarrays were used as two independent methods to study protein-protein interactions. With this combined approach we were able to detect nine calcium-dependent interactions between Arg-Gly-Ser-(RGS)-His6 tagged proteins derived from the library and GST-tagged S100B and S100A6, respectively. For the protein microarray affinity-purified proteins from the expression library were spotted onto modified glass slides and probed with the S100 proteins. SPR experiments were performed in the same setup and in a vice-versa approach reversing analytes and ligands to determine distinct association and dissociation patterns of each positive interaction. Besides already known interaction partners, several novel binders were found independently with both detection methods, albeit analogous immobilization strategies had to be applied in both assays.     

Unbinding forces of single pertussis toxin-antibody complexes measured by atomic force spectroscopy correlate with their dissociation rates determined by surface plasmon resonance

An inactivated form of pertussis toxin (PTX) is the primary component of currently available acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. The PTX analyzed here is purified at industrial scale and is subsequently inactivated using glutaraldehyde. The influence of this treatment on antibody recognition is of crucial importance and is analyzed in this study. Surface plasmon resonance (SPR) experiments using PTX and its inactivated form (toxoid) with 10 different monoclonal antibodies were conducted. PTX was found to recognize the antibodies with an average affinity of 1.34 ± 0.50 nM, and chemical inactivation caused only a modest decrease in affinity by a factor of approximately 4.5. However, glutaraldehyde treatment had contrary effects on the kinetic association constant k(a) and the dissociation constant k(d) . A significant reduction in k(a) was observed, whereas the dissociation of the toxoid from the bound antibody occurred slower than PTX. These data indicate that the chemical inactivation of PTX not only reduces the velocity of antibody recognition but also stabilizes the interaction with antibodies as shown by a reduction in k(d) . The same interactions were also studied by dynamic force spectroscopy (DFS). Data reveal a correlation between the k(d) values determined by SPR and the mean unbinding force as measured by DFS. The unbinding forces of one complex were determined as a function of the loading rate to directly estimate the k(d) value. Several interactions were impossible to be analyzed using SPR because of ultratight binding. Using DFS, the unbinding forces of these interactions were determined, which in turn could be used to estimate k(d) values. The use of DFS as a technique to study ultratight binding is discussed.     

Characterisation of mucosal and systemic immune responses in rainbow trout (Oncorhynchus mykiss) using surface plasmon resonance

Mucosal and systemic antibody production in rainbow trout, Oncorhynchus mykiss (Walbaum), was evaluated following different antigen delivery routes. A BIAcore instrument (Pharmacia) allowed direct detection of antibody-antigen interactions by surface plasmon resonance changes. These interactions were measured in real-time without secondary reagents or extraneous labels. Groups of rainbow trout were immunised with a hapten-carrier antigen consisting of fluorescein isothiocyanate (FITC) conjugated to keyhole limpet haemocyanin (KLH) or phosphate buffered saline (PBS) pH 7.2. Antigens were administered intraperitoneally (i.p.) with or without Freund's complete adjuvant (FCA) or peranally (p.a.) directly to the gastrointestinal (GI) tract. Serum and mucosal anti-FITC responses were significantly (P<0.05) higher in FITC-KLH/FCA groups, clearly showing that adjuvant incorporation enhances mucosal as well as sytemic immunity. Antigen uptake and processing in fish immunised p.a. and i.p. without adjuvant was much less efficient and resulted in relatively low levels of serum and mucosal antibody production. Interestingly, mucosal responses in these groups peaked prior to serum responses suggesting possible early stimulation of mucosal defences. Mucosal antibody production in fish receiving FITC-KLH/FCA correlated more closely with serum responses, indicating possible transfer of serum derived antibody to mucosal sites. Mucosal and serum responses were confirmed as immunoglobulin (Ig) by subsequent reactivity with an anti-trout serum IgM monoclonal antibody (1.14) passed over flow cells containing anti-FITC antibodies. Further analysis showed significantly lower (P<0.05) reactivity of early mucus anti-FITC components (4 weeks post-immunisation) to 1.14. Purified serum and mucus Ig from non-immunised fish showed different protein banding patterns by SDS-PAGE under reducing conditions. Immunoblotting with 1.14 also showed weak reactivity to mucus Ig in control fish while reacting strongly to mucus Ig from immunised fish. These data suggest that early mucosal responses in trout may consist of heterogeneous forms of Ig differing in characteristics to serum Ig. BIAcore analysis in this context and as a means of measuring antibody response proved useful, and has the potential to become a valuable new tool in the study of fish immunology.     

Real time analysis of the RNAI-RNAII-Rop complex by surface plasmon resonance: from a decaying surface to a standard kinetic analysis

RNA loop-loop complexes are motifs that regulate biological functions in both prokaryotic and eukaryotic organisms. In E. coli, RNAI, an antisense RNA encoded by the ColE1 plasmid, regulates the plasmid replication by recognizing through loop-loop interactions RNAII, the RNA primer that binds to the plasmidic DNA to initiate the replication. Rop, a plasmid-encoded homodimeric protein interacts with this transient RNAI-RNAII kissing complex. A surface plasmon resonance (SPR)-based biosensor was used to investigate this protein-nucleic acid ternary complex, at 5 degrees C, in experimental conditions such as the protein binds either to the loop-loop complex while it dissociates or to a saturated stable RNAI-RNAII surface. The results show that RNAI hairpin dissociates from the RNAII surface 110 times slower in the presence of Rop than in its absence. Rop binds to RNAI-RNAII with an on-rate of 3.6 x 10(6) M(-1) s(-1) and an off-rate of 0.11 s(-1), resulting in a binding equilibrium constant equal to 31 nM. A Scatchard-plot analysis of the interaction monitored by SPR confirms a 1:1 complex of Rop and RNAI-RNAII as observed for non-natural Rop-loop-loop complexes.     

Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein

Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP‐Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26–27 kDa). Seventy‐two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty‐two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7–9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease‐like and three thiol protease‐like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca²⁺, Mg²⁺_, Mn²⁺, Ni²⁺, Zn²⁺, Cu²⁺, and Co²⁺ was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti‐MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti‐MBP abzymes, which can attack MBP of myelin‐proteolipid sheath of axons and play an important role in MS and SLE pathogenesis. Copyright © 2015 John Wiley & Sons, Ltd.     

Electron paramagnetic resonance spectroscopy and molecular modelling of the interaction of myelin basic protein (MBP) with calmodulin (CaM)-diversity and conformational adaptability of MBP CaM-targets

The classic 18.5 kDa isoform of murine myelin basic protein (mMBP) has been shown to bind calmodulin (CaM) strongly and specifically in vitro. Here, we have used site-directed spin labelling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy to map more precisely the sites of interaction of recombinant mMBP (rmMBP) with CaM. On the basis of these and previous experimental data, and the predictions of CaM-binding motifs using the Calmodulin Target Database (), three main segments of MBP were suggested for the interaction. The first site is located at the C-terminus; the second one lies in the central portion of the protein and forms an amphipathic alpha-helix in reconstituted myelin-mimetic systems; the third is quite close to the N-terminus. The murine Golli-MBP isoform J37 has also been shown to bind CaM in vitro, and an interaction site was predicted in the N-terminal Golli-specific portion of the protein. From these four segments, we selected peptide fragments of 12-14 residues in length, chosen on the bases of their amphipathicity and CaM-target characteristics. We modelled each of these peptides as alpha-helices, and performed docking simulations to investigate their interactions with the CaM peptide-binding tunnel. Different yet almost equally favourable CaM-binding modes were found for each of them. The experimental SDSL/EPR and theoretical modelling results were in good agreement, and supported the conjecture that there are several plausible CaM-binding sites in MBP, that could be induced into an alpha-helical conformation by their interaction with CaM and account for strong immobilisation of spin-labeled residues in all three segments. Phosphorylation and deimination were also emulated and simulated for known sites of MBP post-translational modification. The results obtained confirmed the appropriate utilisation of simple residue substitutions to mimic the natural modifications, and demonstrated molecular mechanisms by which MBP-CaM interactions could be modulated in vivo.     

How human IgGs against myelin basic protein (MBP) recognize oligopeptides and MBP

Myelin basic protein (MBP) is a major protein of myelin‐proteolipid shell of axons, and it plays an important role in pathogenesis of multiple sclerosis. In the literature, there are no data on how antibodies recognize different protein antigens including MBP. A stepwise increase in ligand complexity was used to estimate the relative contributions of virtually every amino acid residue (AA) of a specific 12‐mer LSRFSWGAEGQK oligopeptide corresponding to immunodominant sequence of MBP to the light chains and to intact anti‐MBP IgGs from sera of patients with multiple sclerosis. It was shown that the minimal ligands of the light chains of IgGs are many different free AAs (K _d = 0.51–0.016 M), and each free AA interacts with the specific subsite of the light chain intended for recognition of this AA in specific LSRFSW oligopeptide. A gradual transition from Leu to LSRFSWGAEGQK leads to an increase in the affinity from 10⁻¹ to 2.3 × 10⁻⁴ M because of additive interactions of the light chain with 6 AAs of this oligopeptide and then the affinity reaches plateau. The contributions of 6 various AAs to the affinity of the oligopeptide are different (K _d, M): 0.71 (S), 0.44 (R), 0.14 (F), 0.17 (S), and 0.62 (W). Affinity of nonspecific oligopeptides to the light chains of IgGs is significantly lower. Intact MBP interacts with both light and heavy chains of IgGs demonstrating 192‐fold higher affinity than the specific oligopeptide. It is a first quantitative analysis of the mechanism of proteins recognition by antibodies. The thermodynamic model was constructed to describe the interactions of IgGs with MBP. The data obtained can be very useful for understanding how antibodies against many different proteins can recognize these proteins.     

Synthetic peptide vaccine development: measurement of polyclonal antibody affinity and cross-reactivity using a new peptide capture and release system for surface plasmon resonance spectroscopy

A method has been developed for measurement of antibody affinity and cross-reactivity by surface plasmon resonance spectroscopy using the EK-coil heterodimeric coiled-coil peptide capture system. This system allows for reversible capture of synthetic peptide ligands on a biosensor chip surface, with the advantage that multiple antibody-antigen interactions can be analyzed using a single biosensor chip. This method has proven useful in the development of a synthetic peptide anti-Pseudomonas aeruginosa (PA) vaccine. Synthetic peptide ligands corresponding to the receptor binding domains of pilin from four strains of PA were conjugated to the E-coil strand of the heterodimeric coiled-coil domain and individually captured on the biosensor chip through dimerization with the immobilized K-coil strand. Polyclonal rabbit IgG raised against pilin epitopes was injected over the sensor chip surface for kinetic analysis of the antigen-antibody interaction. The kinetic rate constants, k(on) and k(off), and equilibrium association and dissociation constants, KA and KD, were calculated. Antibody affinities ranged from 1.14 x 10(-9) to 1.60 x 10(-5) M. The results suggest that the carrier protein and adjuvant used during immunization make a dramatic difference in antibody affinity and cross-reactivity. Antibodies raised against the PA strain K pilin epitope conjugated to keyhole limpet haemocyanin using Freund's adjuvant system were more broadly cross-reactive than antibodies raised against the same epitope conjugated to tetanus toxoid using Adjuvax adjuvant. The method described here is useful for detailed characterization of the interaction of polyclonal antibodies with a panel of synthetic peptide ligands with the objective of obtaining high affinity and cross-reactive antibodies in vaccine development.