Altering drug tolerance of surface plasmon resonance assays for the detection of anti-drug antibodies

Anti-drug antibody (ADA) responses are a concern for both drug efficacy and safety, and high drug concentrations in patient samples may inhibit ADA assays. We evaluated strategies to improve drug tolerance of surface plasmon resonance (SPR) assays that detect ADAs against a bispecific Adnectin drug molecule that consists of an anti-VEGFR2 domain linked to an anti-IGF-1R domain (V-I-Adnectin). Samples containing ADAs against V-I-Adnectin and various drug concentrations were tested in the presence of 1 M guanidine hydrochloride (Gdn), at pH values ranging from 4.5 to 7.4 and temperatures of up to 37 °C. Temperature had a negligible effect in weakening the affinity of interaction of monoclonal antibodies with polyethylene glycol(PEG)–V-I-Adnectin and did not increase drug tolerance of the ADA assay. Low pH increased drug tolerance of the assay relative to pH 7.4 but caused nonspecific binding of the drug during competition experiments. The chaotropic agent Gdn lowered the affinity of interaction between an anti-V-Adnectin monoclonal antibody and the drug (from K_D = 0.93 nM to K_D = 348 nM). That decrease in the affinity of drug–ADA interaction correlated with an increase of assay drug tolerance. Conditions that lower drug–ADA interaction affinity could also be used to develop drug-tolerant SPR assays for other systems.     

Studies on the membrane interactions of the cyclotides kalata B1 and kalata B6 on model membrane systems by surface plasmon resonance

In this study we have demonstrated the interactions of kalata B1 and its naturally occurring analogue kalata B6 with five model lipid membranes and have analyzed the binding kinetics using surface plasmon resonance. Two kalata peptides showed a higher affinity for the phosphatidylethanolamine-containing membranes, indicating that the peptides would bind selectively to bacterial membranes. Also we have optimized the procedure for the immobilization of five liposome mixtures and have shown that the procedure provides reproducible levels of immobilized liposomes and could be used to screen the selective binding of putative antimicrobial peptides to model mammalian or microbial phospholipid membranes.     

Insights into the domains required for dimerization and assembly of human alphaB crystallin

Protein pin array technology was used to identify subunit-subunit interaction sites in the small heat shock protein (sHSP) alphaB crystallin. Subunit-subunit interaction sites were defined as consensus sequences that interacted with both human alphaA crystallin and alphaB crystallin. The human alphaB crystallin protein pin array consisted of contiguous and overlapping peptides, eight amino acids in length, immobilized on pins that were in a 96-well ELISA plate format. The interaction of alphaB crystallin peptides with physiological partner proteins, alphaA crystallin and alphaB crystallin, was detected using antibodies and recorded using spectrophotometric absorbance. Five peptide sequences including 37LFPTSTSLSPFYLRPPSF54 in the N terminus, 75FSVNLDVK82)(beta3), 131LTITSSLS138 (beta8) and 141GVLTVNGP148 (beta9) that form beta strands in the conserved alpha crystallin core domain, and 155PERTIPITREEK166 in the C-terminal extension were identified as subunit-subunit interaction sites in human alphaB crystallin using the novel protein pin array assay. The subunit-subunit interaction sites were mapped to a three-dimensional (3D) homology model of wild-type human alphaB crystallin that was based on the crystal structure of wheat sHSP16.9 and Methanococcus jannaschi sHSP16.5 (Mj sHSP16.5). The subunit-subunit interaction sites identified and mapped onto the homology model were solvent-exposed and had variable secondary structures ranging from beta strands to random coils and short alpha helices. The subunit-subunit interaction sites formed a pattern of hydrophobic patches on the 3D surface of human alphaB crystallin.     

Analysis of Cre-loxP interaction by surface plasmon resonance: influence of spermidine on cooperativity

To study target site selectivity of one important class of DNA-binding proteins, site-specific DNA recombinases, we developed an automated real-time kinetic assay based on surface plasmon resonance (BIACORE) and formulated a curve-fitting model that takes into account cooperative interactions. Monitoring the interaction between the Cre DNA recombinase and its specific target site loxP by BIACORE, we found that Cre associates with loxP tightly and highly cooperatively. We observed that the cooperative moment of the Cre-loxP interaction is strongly dependent on the concentration of spermidine, a small polyamine influencing DNA conformation. Thus, DNA conformation can have a profound impact on substrate recognition and subsequent recombination.     

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.     

Stabilization of oligomeric structure of α‐crystallin by Zn+2 through intersubunit bridging

α‐Crystallin, the major protein of mammalian eye lens, is a member of the small heat shock protein family and is a molecular chaperone. We previously reported that its molecular chaperone function as well as stability increased in presence of Zn⁺². Despite the effect of Zn⁺² on the structure and function of α‐crystallin, evidence for direct interaction between them remained elusive. We now present the MALDI mass spectrometric data that shows direct evidence of Zn⁺² binding to recombinant αA‐ and αB‐crystallin. The binding stoichiometry was over three Zn⁺² per subunit of α‐crystallin at zinc/protein molar ratio of 20. Observation of multiple Zn⁺² binding is consistent with the large increase in thermodynamic stability. Sequence‐based analysis of αA‐ and αB‐crystallin predicted both proteins to be nonzinc binding proteins. Our dynamic light scattering data shows that Zn⁺² stabilizes the oligomeric structure of α‐crystallin by bridging neighboring subunits in multiple centers. Despite the low affinity binding, the intersubunit bridging by multiple Zn⁺² makes the oligomer so stable that oligomer breakdown does not occur even at 6M urea. The subunit bridging has been supported by our FRET data that showed absence of subunit exchange in presence of zinc. MALDI data also showed that the interaction of α‐crystallin with Zn⁺² is quite different from other bivalent metal ions. Bound Zn⁺² could be easily removed by dialysis of the complex. The relevance of such weak interaction on the stability of the oligomeric structure of α‐crystallin and its function in the eye lens has been discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 105–116, 2011.     

NTA-mediated protein capturing strategy in screening experiments for small organic molecules by surface plasmon resonance

Nitrilotriacetate (NTA)-mediated capture of a histidine-tagged protein is widely used as an easy and simple method to reversibly immobilize the protein onto a sensor chip for surface plasmon resonance (SPR). However, in spite of its advantages, the NTA-capturing strategy is rarely employed for ligand screening experiments using SPR, because it was thought to cause substantial errors in binding responses, due to the inevitable protein dissociation during the monitoring period. In this study, as demonstrated in a ligand screening for the histidine-tagged SH3 domain of the human phosphatidylinositol 3-kinase p85alpha subunit, false responses after adhesion of undesirable compounds to a target protein could be minimized with the NTA strategy, while binding responses of a positive control peptide still stayed within a 1%-deviation against the theoretical binding capacity.     

Serum antibody screening by surface plasmon resonance using a natural glycan microarray

A surface plasmon resonance (SPR) based natural glycan microarray was developed for screening of interactions between glycans and carbohydrate-binding proteins (CBPs). The microarray contained 144 glycan samples and allowed the real-time and simultaneous screening for recognition by CBPs without the need of fluorescent labeling. Glycans were released from their natural source and coupled by reductive amination with the fluorescent labels 2-aminobenzamide (2AB) or anthranilic acid (AA) followed by high-performance liquid chromatography (HPLC) fractionation making use of the fluorescent tag. The released and labeled glycans, in addition to fluorescently labeled synthetic glycans and (neo)glycoproteins, were printed on an epoxide-activated chip at fmol amounts. This resulted in covalent immobilization, with the epoxide groups forming covalent bonds to the secondary amine groups present on the fluorescent glycoconjugates. The generated SPR glycan array presented a subset of the glycan repertoire of the human parasite Schistosoma mansoni. In order to demonstrate the usefulness of the array in the simultaneous detection of glycan-specific serum antibodies, the anti-glycan antibody profiles from sera of S. mansoni-infected individuals as well as from non-endemic uninfected controls were recorded. The SPR screening was sensitive for differences between infection sera and control sera, and revealed antibody titers and antibody classes (IgG or IgM). All SPR analyses were performed with a single SPR array chip, which required regeneration and blocking of the chip before the application of a serum sample. Our results indicate that SPR-based arrays constructed from glycans of natural or synthetic origin, pure or as mixture, can be used for determining serum antibody profiles as possible markers for the infection status of an individual.     

Strategies for the identification and purification of ligands for orphan biomolecules

The isolation of related genes with evolutionary conserved motifs by the application ofpolymerase chain reaction-based molecular biology techniques, or from database searchingstrategies, has facilitated the identification of new members of protein families. Many of theseprotein molecules will be involved in protein–protein interactions (e.g. growth factors,receptors, adhesion molecules), since such interactions are intrinsic to virtually every cellularprocess. However, the precise biological function and specific binding partners of these novelproteins are frequently unknown, hence they are known as orphan molecules.Complementary technologies are required for the identification of the specific ligands orreceptors for these and other orphan proteins (e.g., antibodies raised against crude biologicalextracts or whole cells). We describe herein several alternative strategies for the identification,purification and characterisation of orphan peptide and protein molecules, specifically thesynergistic use of micropreparative HPLC and biosensor techniques.     

Single-cycle kinetic analysis of ternary DNA complexes by surface plasmon resonance on a decaying surface

Complexes involving three DNA strands were used to demonstrate that the single-cycle kinetics (SCK) method, which consists in injecting sequentially samples at increasing concentrations and until now used exclusively to investigate bimolecular complexes by surface plasmon resonance, can be extended to the kinetic analysis of ternary complexes. DNA targets, B, were designed with sequences of variable lengths on their 3' sides that recognise a surface-immobilized biotinylated DNA anchor, A. These targets displayed on their 5' sides sequences that recognise DNA oligonucleotides of variable lengths, C, namely the analytes. Combinations of B and C DNA oligonucleotides on A generated ternary complexes each composed of two Watson-Crick helices displaying different kinetic properties. The target-analyte B-C duplexes were formed by sequentially injecting three increasing concentrations of the analytes C during the dissociation phase of the target B from the anchor A. The sensorgrams for the target-analyte complexes dissociating from the functionalized surface were successfully fitted by the SCK method while the target dissociated from the anchor, i.e. on a decaying surface. Within the range of applicability of the method which is driven by the rate of dissociation of the target from the anchor, the rate and equilibrium constants characteristic of these target-analyte duplexes of the ternary complexes did not depend on how fast the targets dissociated from the immobilized DNA anchor. In addition the results agreed very well with those obtained when such duplexes were analysed directly as bimolecular complexes, i.e. when the target, modified with a biotin, was directly immobilized onto a streptavidin sensor chip surface rather than captured by an anchor. Therefore the method we named SCKODS (Single-Cycle Kinetics On a Decaying Surface) can also be used to investigate complexes formed during a dissociation phase, in a ternary complex context. The SCKODS method can be combined with the SCK one to fully characterize the two bimolecular complexes of a ternary complex.     

Direct analysis of a GPCR-agonist interaction by surface plasmon resonance

Despite their clinical importance, detailed analysis of ligand binding at G-protein coupled receptors (GPCRs) has proved difficult. Here we successfully measure the binding of a GPCR, neurotensin receptor-1 (NTS-1), to its ligand, neurotensin (NT), using surface plasmon resonance (SPR). Specific responses were observed between NT and purified, detergent-solublised, recombinant NTS-1, using a novel configuration where the biotinylated NT ligand was immobilised on the biosensor surface. This SPR approach shows promise as a generic approach for the study of ligand interactions with other suitable GPCRs.     

Evidence for specific subunit distribution and interactions in the quaternary structure of α‐crystallin

The quaternary structure of α‐crystallin is dynamic, a property which has thwarted crystallographic efforts towards structural characterization. In this study, we have used collision‐induced dissociation mass spectrometry to examine the architecture of the polydisperse assemblies of α‐crystallin. For total α‐crystallin isolated directly from fetal calf lens using size‐based chromatography, the αB‐crystallin subunit was found to be preferentially dissociated from the oligomers, despite being significantly less abundant overall than the αA‐crystallin subunits. Furthermore, upon mixing molar equivalents of purified αA‐ and αB‐crystallin, the levels of their dissociation were found to decrease and increase, respectively, with time. Interestingly though, dissociation of subunits from the αA‐ and αB‐crystallin homo‐oligomers was comparable, indicating that strength of the αA:αA, and αB:αB subunit interactions are similar. Taken together, these data suggest that the differences in the number of subunit contacts in the mixed assemblies give rise to the disproportionate dissociation of αB‐crystallin subunits. Limited proteolysis mass spectrometry was also used to examine changes in protease accessibility during subunit exchange. The C‐terminus of αA‐crystallin was more susceptible to proteolytic attack in homo‐oligomers than that of αB‐crystallin. As subunit exchange proceeded, proteolysis of the αA‐crystallin C‐terminus increased, indicating that in the hetero‐oligomeric form this tertiary motif is more exposed to solvent. These data were used to propose a refined arrangement for the interactions of the α‐crystallin domains and C‐terminal extensions of subunits within the α‐crystallin assembly. In particular, we propose that the palindromic IPI motif of αB‐crystallin gives rise to two orientations of the C‐terminus. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Heat shock factor 1 deficiency via its downstream target gene αB‐crystallin (Hspb5) impairs p53 degradation

Heat shock factor Hsf1 regulates the stress‐inducibility of heat shock proteins (Hsps) or molecular chaperones. One of the functions attributed to Hsps is their participation in folding and degradation of proteins. We recently showed that hsf1^?/? cells accumulate ubiquitinated proteins. However, a direct role for Hsf1 in stability of specific proteins such as p53 has not been elucidated. We present evidence that cells deficient in hsf1 accumulate wild‐type p53 protein. We further show that hsf1^?/? cells express lower levels of αB‐crystallin and cells deficient in αB‐crystallin also accumulate p53 protein. Reports indicate that αB‐crystallin binds to Fbx4 ubiquitin ligase, and they target cyclin D1 for degradation through a pathway involving the SCF (Skp1‐Cul1‐F‐box) complex. Towards determining a mechanism for p53 degradation involving αB‐crystallin and Hsf1, we have found that ectopic expression of Fbx4 in wild‐type mouse embryo fibroblasts (MEFs) expressing mutant p53 (p53R175H) leads to increase in its degradation, while MEFs deficient in hsf1 or αBcry are defective in degradation of this p53 protein. In addition, immunoprecipitated p53R175H from wild‐type MEFs is able to pull‐down both αB‐crystallin and Fbx4. Finally, immunoprecipitated wild‐type p53 from doxorubicin treated U2OS cells can pull‐down endogenous αB‐crystallin and Fbx4. These results indicate that hsf1‐ and αBcry‐deficient cells accumulate p53 due to reduced levels of αB‐crystallin in these cells. Elevated levels of p53 in hsf1‐ and αBcry‐deficient cells lead to their increased sensitivity to DNA damaging agents. These data reveal a novel mechanism for protein degradation through Hsf1 and αB‐crystallin. J. Cell. Biochem. 107: 504–515, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor–UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5 ng/ml. The detection limit is 0.06 ng/ml for the biosensor with antibody and 0.08 ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20 ng/ml for plasma samples and 0.30 to 0.49 ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.     

IgG subclass specificity to C1q determined by surface plasmon resonance using Protein L capture technique

Recombinant monoclonal antibodies (mAbs) have become an important category of biological therapeutics. mAbs share the same structures and biological functions as endogenous IgG molecules. One function is complement-dependent cytotoxicity (CDC) initiation by binding of C1q. Traditionally, ELISA methods have been utilized to measure C1q binding. A new robust capture method was established in this study to measure the binding affinity of C1q to antibodies by surface plasmon resonance (SPR). The utility of this method was demonstrated by determination of the difference in IgG subclass specificity of C1q binding.     

Chiral recognition of dextran sulfate with D- and L-cystine studied by multiwavelength surface plasmon resonance

A multiwavelength surface plasmon resonance (mwSPR) approach has been developed to study the chiral discrimination between D- and L-cystine (Cys). A monolayer of the two enantiomers was separately assembled on a pair of gold films of about 50 nm in thickness and their resonance wavelength shifts, Deltalambda, were measured under a continuous flow of an identical chiral probe solution. Dextran sulfate (DS) was found to be an excellent chiral probe because it has rich chiral centers and is large enough to produce sensitive mwSPR response. The chiral discrimination was investigated either by Deltalambda(max), the maximum resonance wavelength shift in recognition equilibrium, or by recognition kinetics (Deltalambda vs time). The equilibrium data showed that D-Cys yielded always the smaller Deltalambda(max) as compared to L-Cys at pH 5.0 or above. This differentiation was enlarged by raising the probe content and became naught at pH <4.5. The kinetic results showed that, as pH increased from 5.0 to 7.5, the non-equilibrium Deltalambda for D-Cys rose above the level for L-Cys at the first 30s of recognition but came back gradually to its equilibrium position after about 150 s, with crossing at 50--150 s depending on DS concentration. This phenomenon was thought to be the result of molecular orientation adjustment after DS binding to D-Cys. Both kinetic and thermodynamic mechanisms were thus considered to be deeply involved in the investigated chiral recognition system.     

Binding determinants of the small heat shock protein, αB‐crystallin: recognition of the ‘IxI’ motif

Small heat shock proteins (sHSPs) play a central role in protein homeostasis under conditions of stress by binding partly unfolded, aggregate‐prone proteins and keeping them soluble. Like many sHSPs, the widely expressed human sHSP, αB‐crystallin (‘αB’), forms large polydisperse multimeric assemblies. Molecular interactions involved in both sHSP function and oligomer formation remain to be delineated. A growing database of structural information reveals that a central conserved α‐crystallin domain (ACD) forms dimeric building blocks, while flanking N‐ and C‐termini direct the formation of larger sHSP oligomers. The most commonly observed inter‐subunit interaction involves a highly conserved C‐terminal ‘IxI/V’ motif and a groove in the ACD that is also implicated in client binding. To investigate the inherent properties of this interaction, peptides mimicking the IxI/V motif of αB and other human sHSPs were tested for binding to dimeric αB‐ACD. IxI‐mimicking peptides bind the isolated ACD at 22°C in a manner similar to interactions observed in the oligomer at low temperature, confirming these interactions are likely to exist in functional αB oligomers.