Surface Plasmon Resonance Methodology Research Articles

Label-Free Analysis of Binding and Inhibition of SARS-Cov-19 Spike Proteins to ACE2 Receptor with ACE2-Derived Peptides by Surface Plasmon Resonance.

SARS-CoV-2 has been shown to enter and infect human cells via interactions between spike protein (S glycoprotein) and angiotensin-converting enzyme 2 (ACE2). As such, it may be possible to suppress the infection of the virus via the blocking of this binding interaction through the use of specific peptides that can mimic the human ACE 2 peptidase domain (PD) α 1-helix. Herein, we report the use of competitive assays along with surface plasmon resonance (SPR) to investigate the effect of peptide sequence and length on spike protein inhibition. The characterization of these binding interactions helps us understand the mechanisms behind peptide-based viral blockage and develop SPR methodologies to quickly screen disease inhibitors. This work not only helps further our understanding of the important biological interactions involved in viral inhibition but will also aid in future studies that focus on the development of therapeutics and drug options. Two peptides of different sequence lengths, [30-42] and [22-44], based on the α 1-helix of ACE2 PD were selected for this fundamental investigation. In addition to characterizing their inhibitory behavior, we also identified the critical amino acid residues of the RBD/ACE2-derived peptides by combining experimental results and molecular docking modeling. While both investigated peptides were found to effectively block the RBD residues known to bind to ACE2 PD, our investigation showed that the shorter peptide was able to reach a maximal inhibition at lower concentrations. These inhibition results matched with molecular docking models and indicated that peptide length and composition are key in the development of an effective peptide for inhibiting biophysical interactions. The work presented here emphasizes the importance of inhibition screening and modeling, as longer peptides are not always more effective.

Multivalent Interactions with Intrinsically Disordered Proteins Probed by Surface Plasmon Resonance.

Multivalent interactions underpin associations between intrinsically disordered proteins (IDPs) and their binding partners. This is a subject of considerable interest and governs how nuclear transport receptors (NTRs) orchestrate the nucleocytoplasmic transport (NCT) of signal-specific cargoes through nuclear pore complexes (NPCs) in eukaryotic cells. Specifically, IDPs termed phenylalanine-glycine nucleoporins (FG Nups) exert multivalent interactions with NTRs to facilitate their transport selectivity and speed through the NPC. Here, we document the use of surface plasmon resonance (SPR) to quantify the affinity and kinetics of NTR-FG Nup binding as a function of FG Nup surface density. Moreover, we describe an in situ method that measures conformational height changes that occur in a FG Nup layer following NTR-binding. Protocols by which the as-obtained SPR results are treated with respect to mass transport limitations are further described. Overall, the SPR methodology described here can be applied to studying multivalent interactions and the role of avidity in diverse biological and biointerfacial systems.

Novel reagents for human prolactin research: large-scale preparation and characterization of prolactin receptor extracellular domain, non-pegylated and pegylated prolactin and prolactin receptor antagonist.

To provide new tools for in vitro and in vivo prolactin (PRL) research, novel protocols for large-scale preparation of untagged human PRL (hPRL), a hPRL antagonist (del 1-9-G129R hPRL) that acts as a pure antagonist of hPRL in binding to hPRL receptor extracellular domain (hPRLR-ECD), and hPRLR-ECD are demonstrated. The interaction of del 1-9-G129R hPRL with hPRLR-ECD was demonstrated by competitive non-radioactive binding assay using biotinylated hPRL as the ligand and hPRLR-ECD as the receptor, by formation of stable 1:1 complexes with hPRLR-ECD under non-denaturing conditions using size-exclusion chromatography, and by surface plasmon resonance methodology. In all three types of experiments, the interaction of del 1-9-G129R hPRL was equal to that of unmodified hPRL. Del 1-9-G129R hPRL inhibited the hPRL-induced proliferation of Baf/LP cells stably expressing hPRLR. Overall, the biological properties of del 1-9-G129R hPRL prepared by the protocol described herein were similar to those of the antagonist prepared using the protocol reported in the original study; however, the newly described protocol improved yields by >6-fold. To provide long-lasting hPRL as a new reagent needed for in vivo experiments, we prepared its mono-pegylated analogue and found that pegylation lowers its biological activity in a homologous in vitro assay. As its future use will require the development of a PRL antagonist with highly elevated affinity, del 1-9-G129R hPRL was expressed on the surface of yeast cells. It retained its binding capacity for hPRLR-ECD, and this methodology was shown to be suitable for future development of high-affinity hPRL antagonists using a library of randomly mutated open reading frame of del 1-9-G129R hPRL and selecting high-affinity mutants by yeast surface display methodology.

Metal Nanoparticles for the Treatment and Diagnosis of Neurodegenerative Brain Diseases.

This review focuses on the application of metal nanoparticles in the diagnosis and treatment of Alzheimer's and Parkinson's diseases. Metal nanoparticles present interesting physicochemical properties that can be applied to increase biomarker detection sensitivities in vitro and in vivo. Furthermore, these nanoparticles could be used in different strategies for the treatment of central nervous system diseases, particularly in regards to drug delivery. Herein, specific potential applications of metal nanoparticles are separately discussed for the contexts of in vitro diagnoses and treatments. Briefly, research using surface plasmon resonance methodologies has mainly used these nanoparticles for the in vitro detection of Aβ and, to a lesser extent, of α-synuclein. Regarding treatment approaches, in vitro studies have focused on using metal nanoparticles to manipulate the Aβ aggregation, thus reducing toxicity. Furthermore, in vivo applications of metal nanoparticles are also discussed, with many of the existing studies focusing on a magnetic nanoparticle-detection of Aβ through magnetic resonance imaging and, to a lesser degree, extension fluorescence techniques. Finally, conclusions and perspectives are provided regarding the real potential for using metal nanoparticles in the treatment and diagnosis of central nervous system diseases.

Quantification and simultaneous evaluation of the bioactivity of antibody produced in CHO cell culture-The use of the ectodomain of FcγRI and surface plasmon resonance-based biosensor.

A surface plasmon resonance (SPR)-based assay has been developed in order to quantify the monoclonal antibody (Mab) Trastuzumab within the supernatant of a mammalian cell culture using the ectodomain of FcγRI (CD64) and confirm Mab bioactivity, i.e. binding to its antigen Her2, in a single biosensing experiment. Under partial mass transport limitation, we were able to quantify Mab present in unpurified samples taken throughout the cell culture. While Mab capture on the biosensor surface confirmed the ability of its Fc region to bind to FcγRI, the binding activity of its Fab region was also tested by injecting increasing concentrations of the Mab antigen (Her2). The kinetics of the interactions we recorded from 48h post transfection until the end of the culture, were superimposable, which highly suggested that the quality attributes of the antibody were conserved throughout the process. This SPR methodology is thus of great interest for atline quality control analysis during Mab production campaign.

Surface plasmon resonance methodology for monitoring polymerization kinetics and morphology changes of brushes—evaluated with poly(N-isopropylacrylamide)

Polymerization from surfaces and the resulting “brushes” have many uses in the development of novel materials and functional interfaces. However, it is difficult to accurately monitor the polymerization rate, which limits the use of polymer brushes in applications where control of thickness is desirable. We present a new methodology based on angular surface plasmon resonance (SPR) which provides real-time measurements of the thickness evolution during atom transfer radical polymerization, using poly(N-isopropylacrylamide) as an example. Our data analysis shows that the growth is linear with a rate of ∼20nm/min in a water/methanol mixture up to ∼100nm after which chain termination gradually reduces the growth rate. Further, we introduce an improved method in SPR which makes it possible to determine changes in brush height and refractive index during switching of responsive polymers. The ratio between heights in the coil to globule transition at 32°C in water was found to be almost 5, independent of the initial absolute height up to ∼200nm, in agreement with theory. Complementary quartz crystal microbalance and atomic force microscopy data confirm the accuracy of our results. With the methodology presented here the established SPR technique can be used for quantitative characterization of surface-initiated polymerization and responsive polymer brushes.

Surface plasmon resonance biosensor assay for the analysis of small-molecule inhibitor binding to human and parasitic phosphodiesterases

In the past decade, surface plasmon resonance (SPR) biosensor-based technology has been exploited more and more to characterize the interaction between drug targets and small-molecule modulators. Here, we report the successful application of SPR methodology for the analysis of small-molecule binding to two therapeutically relevant cAMP phosphodiesterases (PDEs), Trypanosoma brucei PDEB1 which is implicated in African sleeping sickness and human PDE4D which is implicated in a plethora of disease conditions including inflammatory pulmonary disorders such as asthma, chronic obstructive pulmonary disease and central nervous system (CNS) disorders. A protocol combining the use of directed capture using His-tagged PDE_CDs with covalent attachment to the SPR surface was developed. This methodology allows the determination of the binding kinetics of small-molecule PDE inhibitors and also allows testing their specificity for the two PDEs. The SPR-based assay could serve as a technology platform for the development of highly specific and high-affinity PDE inhibitors, accelerating drug discovery processes.

Surface plasmon resonance characterization of monoclonal and polyclonal antibodies of malaria for biosensor applications

Surface plasmon resonance (SPR) screening of monoclonal and polyclonal antibodies of Plasmodium falciparum (MoabPf and PoabPf) for recombinant Histidine rich protein-II antigen (Ag) of Pf (rHRP-II Ag) was conducted in a real-time and label-free manner to select an appropriate antibody (Ab) for biosensor applications. In this study 4-mercaptobenzoic acid (4-MBA) modified gold SPR chip was used for immobilizing the Ag and then Ab was interacted. SEM image showed modification of SPR chip with 4-MBA and EDAX confirmed the presence of 4-MBA on the SPR chip. Equilibrium constant (KD) and maximum binding capacity of analyte (Bmax) values for the interaction of MoabPf or PoabPf with the immobilized rHRP-II Ag were calculated and found to be 0.517nM and 48.61m° for MoabPf and 2.288nM and 46.80m° for PoabPf, respectively. In addition, thermodynamic parameters such as ΔG, ΔH and ΔS were determined for the interaction between rHRP-II Ag and MoabPf or PoabPf and the values revealed that the interaction is spontaneous, exothermic and driven by entropy. The kinetics and thermodymanic results of this study revealed that the interaction between MoabPf and rHRP-II Ag is more effective than that of PoabPf due to the fact that MoabPf was derived from a single epitope (single clone) whereas the PoabPf was from the mixture of a number of epitopes (polyclones). Finally, SPR methodology was developed for the sensing of malarial antibodies. The limit of detection was found to be 5.6pg with MoabPf which was found to be the best in our study.

Real-Time Quantification of Proteins Secreted by Artificial Connective Tissue Made from Uni- Or Multidirectional Collagen I Scaffolds and Oral Mucosa Fibroblasts

Previously, we found that oral autologous artificial connective tissue (AACT) had a different protein secretion profile to that of clot-embedded AACT. Other oral mucosa substitutes, having different cell types and scaffolds, had dissimilar secretion profiles of proteins (including that for AACT) that influence healing outcome; thus, to ascertain the profiles of factors secreted by artificial tissue and whether they are influenced by their microstructure might help in understanding their bioactivity. An important component of tissue microstructure is the fiber orientation of the scaffold used for manufacturing it. This work developed a surface plasmon resonance (SPR) methodology to quantify factors secreted by oral artificial connective tissue (ACT) in culture medium, and a method to manufacture unidirectional laminar collagen I scaffolds. The SPR methodology was used for assessing differences in the protein secretion profile of ACT made with collagen scaffolds having different fiber orientation (unidirectional vs multidirectional). Oral fibroblasts seeded onto unidirectional scaffolds increased the secretion of six factors involved in modulating healing compared to those seeded onto multidirectional scaffolds. Histological analysis of uni- and multidirectional ACT showed that cells differ in their alignment and morphology. This SPR-methodology led to nanoscale detection of paracrine factors and might be useful to study biomarkers of three-dimensional cell growth, cell differentiation, and wound-healing progression.

Subunit and small-molecule interaction of ribonucleotide reductases via surface plasmon resonance biosensor analyses

Ribonucleotide reductase (RNR) synthesizes deoxyribonucleotides for DNA replication and repair and is controlled by sophisticated allosteric regulation involving differential affinity of nucleotides for regulatory sites. We have developed a robust and sensitive method for coupling biotinylated RNRs to surface plasmon resonance streptavidin biosensor chips via a 30.5 Å linker. In comprehensive studies on three RNRs effector nucleotides strengthened holoenzyme interactions, whereas substrate had no effect on subunit interactions. The RNRs differed in their response to the negative allosteric effector dATP that binds to an ATP-cone domain. A tight RNR complex was formed in Escherichia coli class Ia RNR with a functional ATP cone. No strengthening of subunit interactions was observed in the class Ib RNR from the human pathogen Bacillus anthracis that lacks the ATP cone. A moderate strengthening was seen in the atypical Aeromonas hydrophila phage 1 class Ia RNR that has a split catalytic subunit and a non-functional ATP cone with remnant dATP-mediated regulatory features. We also successfully immobilized a functional catalytic NrdA subunit of the E.coli enzyme, facilitating study of nucleotide interactions. Our surface plasmon resonance methodology has the potential to provide biological insight into nucleotide-mediated regulation of any RNR, and can be used for high-throughput screening of potential RNR inhibitors.

Evaluation of Surface Plasmon Resonance Relative to High Pressure Liquid Chromatography for the Determination of Paralytic Shellfish Toxins

A surface plasmon resonance (SPR) method, incorporating monoclonal and polyclonal antibodies, was compared to HPLC fluorescence for the determination of paralytic shellfish toxins (PSTs) in shellfish collected from different regions of Canada (n = 33) and Europe (n = 55). Cross-reactivity between saxitoxin (STX) and its structural analogues was determined for both monoclonal (GT-13A) and polyclonal (R895) antibodies. Method detection limits based on IC(10) values, using the SPR methodology (0.55-71.3 ng/mL), in particular for GT-13A, were somewhat higher than those determined using HPLC (0.16-1.29 ng/mL). SPR analyses generally resulted in higher PST levels relative to those obtained using HPLC, although neither antibody successfully responded to the N-1-hydroxylated analogues (e.g., neosaxitoxin). Five and 10 (R895 and GT-13A, respectively) of the 88 samples tested resulted in PST concentrations above the regulatory limit (80 microg/100 g shellfish tissue as STX equivalents), although HPLC responses indicated that these samples were within acceptable levels. Two and five samples were found to have PST concentrations below the regulatory limit using the GT-13A and R895, respectively, when HPLC results exceeded the limit. SPR may be applicable as a screening technique, although improved antibody response to the N-1-hydroxylated PSTs is required prior to this method being safely used for routine testing.

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

A new approach for analyzing lipid–lipid transfer protein interactions is described. The transfer protein is genetically engineered for expression with a C-terminal biotinylated peptide extension (AviTag). This allows protein anchoring to a streptavidin-coated chip for surface plasmon resonance (SPR)-based assessment of lipid binding. Sterol carrier protein-2 (SCP-2), involved in the intracellular trafficking of cholesterol, fatty acids, and other lipids, was selected as the prototype. Biotinylated SCP-2 (bSCP-2) was expressed in Escherichia coli, purified to homogeneity by mutated streptavidin (SoftLink) affinity chromatography, and confirmed by mass spectrometry to contain one biotin group at the expected position. Intermembrane [ 14C]cholesterol transfer was strongly enhanced by bSCP-2, demonstrating that it was functional. Using bSCP-2 immobilized on a Biacore streptavidin chip, we determined on- and off-rate constants along with equilibrium dissociation constants for the following analytes: oleic acid, linoleic acid, cholesterol, and fluorophore (NBD)-derivatized cholesterol. The dissociation constant for NBD–cholesterol was similar to that determined by fluorescence titration for SCP-2 in solution, thereby validating the SPR approach. This method can be readily adapted to other transfer proteins and has several advantages over existing techniques for measuring lipid binding, including (i) the ability to study lipids in their natural states (i.e., without relatively large reporter groups) and (ii) the ability to measure on- and off- rate constants as well as equilibrium constants.

Selection, Characterization, and CDR Shuffling of Naive Llama Single-Domain Antibodies Selected against Auxin and Their Cross-Reactivity with Auxinic Herbicides from Four Chemical Families

Indoleacetic acid (IAA)-binding single-domain antibodies (sdAbs) were isolated from a naive phage-display library constructed from the heavy chain antibody repertoire of a Ilama. The highest-affinity sdAb isolated (CSF2A) had a K(D) of 5-20 microM for two IAA-protein conjugates and a K(D) of 20 microM for free IAA. This sdAb also bound to a synthetic auxin analogue, 1-naphthaleneacetic acid (NAA), and to six auxinic herbicides (K(D) values of 0.5-2 mM), but not to serotonin and tryptophan, which are structurally similar to IAA but have no auxinic activity. To understand how sdAb CSF2A binds IAA and to determine which complementary-determining region(s) (CDR) participate(s) most in binding IAA, CSF2A was shuffled with four other sdAb clones by staggered extension process (StEP). After panning against IAA, two shuffled sdAbs were found: sdAb CSB1A, which originated from three different parental clones, and sdAb CSE8A, derived from two parental clones. These shuffled sdAbs and CSF2A were each fused to the B subunit of the Escherichia coli verotoxin, resulting in the formation of the pentamerized sdAbs V2NCSB1A, V2NCSE8A, and V2NCSF2A, which were analyzed by surface plasmon resonance (SPR) along with the sdAbs previously isolated. The shuffled clones had affinity for IAA (20 microM) similar to that of the highest affinity parental clone CSF2A, but much lower affinity for the auxinic herbicides. CDR2 was instrumental in binding IAA, whereas hydrophobic CDR3 was important for binding the auxinic herbicides. A novel SPR methodology is also described for specific immobilization of pentamerized sdAbs, allowing determination of K(D) values of Ab interaction with underivatized, low molecular weight haptens.

Lipid and Lipid Hydroperoxide Interaction with Sterol Carrier Protein‐2 as Assessed by Surface Plasmon Resonance Methodology

Sterol carrier protein-2 (SCP-2), also known as non-specific lipid transfer protein, plays a crucial role in intracellular lipid trafficking and metabolism. Numerous studies have shown that SCP-2 greatly enhances the translocation rate of cholesterol (Ch) and various phospholipids (PL) between membrane bilayers. Our group recently showed that human recombinant SCP-2 (13.2 kDa) can also accelerate intermembrane transfer of Ch hydroperoxide and PL hydroperoxide species. Whether SCP-2 actually binds Ch or PLs during facilitated translocation has been controversial. The interaction has been assessed by others, using fluorescence titration techniques with fluorophore-tagged Ch or PLs. However, reported binding constants vary widely for any given lipid (e.g. up to 100-fold for Ch), and could be suspect due to the relatively massive attached fluorophore group. To circumvent these problems, we have devised a novel approach for measuring binding, based on genetically appending a biotinylated peptide to the SCP-2 C-terminus for use in surface plasmon resonance (SPR) measurements. Biotinylated SCP-2 was immobilized on a streptavidin-coated chip and the on-rates, off-rates and binding constants for various lipids, including oleic acid, linoleic acid, and the 5α-, 6α-, and 7α-hydroperoxides of Ch were assessed by SPR, using Biacore™ instrumentation. These studies demonstrate that the SPR approach is not only practicable, but clearly superior to previously used approaches. Assessing SCP-2 binding of lipid hydroperoxides by this technique is highly significant in the context of cell damaging oxidative stress because SCP-2 trafficking of these species under stress conditions could greatly expand their range of prooxidant and stress signaling activity. (Supported by NIH grant CA72630)

Direct quantification of the modulation of interaction between cell- or surface-bound LFA-1 and ICAM-1

The functional activity of leukocyte integrins is highly regulated by several mechanisms related to intrinsic molecular properties and receptor interaction with the cell membrane. Here, we present a microkinetic study of the lymphocyte function-associated antigen-1-mediated interaction between flowing Jurkat cells and surface- or cell-bound intercellular adhesion molecule-1 (ICAM-1). We conclude that adhesion is initiated by the formation of a single bond with approximately 0.3 s(-1) dissociation rate, and attachment is subsequently strengthened by the formation of additional bonds during the next 10 s; exposing cells to Mg2+ or Mn2+ resulted in up to a 16-fold increase of the binding frequency, in line with reported measurements performed on isolated molecules with surface plasmon resonance methodology; cell-bound ICAM-1 molecules were more efficient in mediating adhesion than Fc-ICAM-1, properly oriented and bound by surface-adsorbed protein A; and quantitative analysis of binding frequency suggested that adhesion efficiency was ten- to 100-fold lower than the maximum value allowed by previously determined association rates of soluble molecules. It is concluded that the presented methodology provides a simple and unique way of dissecting the initial step of cell adhesion and discriminating between affinity and avidity modulation of adhesion receptors.

Subcloning, Expression, Purification, and Characterization of Recombinant Human Leptin-binding Domain

A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.

Functional promiscuity of squirrel monkey growth hormone receptor toward both primate and nonprimate growth hormones.

Primate growth hormone (GH) has evolved rapidly, having undergone approximately 30% amino acid substitutions from the inferred ancestral eutherian sequence. Nevertheless, human growth hormone (hGH) is physiologically effective when administered to nonprimate mammals. In contrast, its functional counterpart, the human growth hormone receptor (hGHR), has evolved species specificity so that it responds only to Old World primate GHs. It has been proposed that this species specificity of the hGHR is largely caused by the Leu --> Arg change at position 43 after a prior His --> Asp change at position 171 of the GH. Sequence analyses supported this hypothesis and revealed that the transitional phase in the GH:GHR coevolution still persists in New World monkeys. For example, although the GH of the squirrel monkey has the His --> Asp substitution at position 171, residue 43 of its GHR is a Leu, the nonprimate residue. If the squirrel monkey truly represents an intermediate stage of GH:GHR coevolution, its GHR should respond to both hGH and nonprimate GH. Also, if the emergence of species specificity was a result of the selection for a more efficient GH:GHR interaction, then changing residue 43 of the squirrel monkey growth hormone receptor (smGHR) to Arg should increase its binding affinity toward higher primate GH. To test these hypotheses, we performed protein-binding assays between the smGHR and both human and rat GHs, using the surface plasmon resonance methodology. Furthermore, the effects of reciprocal mutations at position 43 of human and squirrel monkey GHRs are measured for their binding affinities toward human and squirrel monkey GHs. The results from the binding kinetic assays clearly demonstrate that the smGHR is in the intermediate state of the evolution of species specificity. Interestingly, the altered residue Arg at position 43 of the smGHR does not lead to an increased binding affinity. The implications of these results on the evolution of the GH:GHR interaction and on functional evolution are discussed.

Calpain Cleavage Promotes Talin Binding to the β3Integrin Cytoplasmic Domain

Talin links integrin beta cytoplasmic domains to the actin cytoskeleton and is involved in the clustering and activation of these receptors. To understand how talin recognizes integrin beta cytoplasmic domains, we configured surface plasmon resonance methodology to measure the interaction of talin with the beta3 integrin cytoplasmic domain. Here we report that the N-terminal approximately 47-kDa talin head domain (talin-H) has a 6-fold higher binding affinity than intact talin for the beta3 tail. The affinity difference is mainly due to a difference in k(on). Calpain cleavage of intact talin released talin-H and resulted in a 16-fold increase in apparent K(a) and a 100-fold increase in apparent k(on). The increase in talin binding after cleavage was greater than predicted for stoichiometric liberation of free talin-H. This additional increase in binding was due to cooperative binding of talin-H and talin rod domain to the beta3 tail. Talin resembles ERM (ezrin, radixin, moesin) proteins in possessing an N-terminal FERM (band four-point-one, ezrin, radixin, moesin) domain. These data show that the talin FERM domain, like that in the ERM proteins, is masked in the intact molecule. Furthermore, they suggest that talin cleavage by calpain may contribute to the effects of the protease on the clustering and activation of integrins.

The Influence of Signal Noise on the Accuracy of Kinetic Constants Measured by Surface Plasmon Resonance Experiments

An analysis is carried out to investigate the accuracy of kinetic parameters obtained using surface plasmon resonance methodology with a BIAcore instrument. The Cramer Rao lower bound for the least possible variance of an estimator of the kinetic parameters is determined. Using simulations it is shown that the standard least-squares estimation technique provides estimates that achieve this bound. The theoretical and simulation results are compared with experimental data obtained from an analysis of the interaction of the myc peptide with the anti-myc antibody, 9E10. This investigation indicates that the accuracy of the results depends on the signal level which has particular relevance to the design of experiments with low signal levels. It is shown how the accuracy of the estimates of the kinetic constants depends on the kinetic constants themselves and how the accuracy of the association constants depends on the concentration of the analyte that is used in the experiment. In addition, the effects of increasing the number of data points in the analysis of dissociation data on the accuracy of the estimates are quantitated. It is also demonstrated that signal averaging of data derived from repeat sensorgrams can result in a significant decrease in the standard deviation of the estimates.

Measurement of Clostridium perfringens β-toxin production by surface plasmon resonance immunoassay

A rapid and sensitive assay using surface plasmon resonance (SPR) immunoassay has been developed for the detection of Clostridium perfringens β-toxin. The SPR immunoassay was conducted off-line by passing fermentation broth by a sensor chip coated with a monoclonal antibody specific for C. perfringens β-toxin. Quantitation of toxin using SPR immunoassay was achieved by mass transport analysis; results were obtained within 20 min. The SPR immunoassay was compared with an ELISA and the traditional bioassay for C. perfringens β-toxin. The SPR immunoassay and ELISA detected at least twofold differences in toxin levels at 95% confidence over a broad range of toxin concentrations. The traditional bioassay did not produce the resolution observed with the immunoassays. The SPR immunoassay allows for real-time monitoring of β-toxin accumulation during production and permits the bioengineer to harvest C. perfringens fermentations when toxin is most concentrated. The SPR methodology may be applied to other fermentations to enhance and optimize toxin yields.