Affinity Measurement Using Surface Plasmon Resonance

Karlsson, Robert; Larsson, Anita

doi:10.1385/1-59259-666-5:389

Cited by 20 publications

(27 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The analysis of the impact of higherorder oligomerization of MBL 3 failed to reveal any functional difference between the oligomers, with only an ∼2-fold difference in binding kinetics and equilibrium constants reported for human 33MBL 3 and 43MBL 3 (18). The standard model for analyzing molecular interactions by SPR is based on the assumption of a simple 1:1 first-order reaction (27,28), often referred to as a pseudo first-order reaction, because the level of complex formation between the molecules in the fluid phase (the analyte) and the surface-exposed ligands is regulated by the concentration of only one of the reactants (i.e., the analyte) (29). As demonstrated by Svitel et al (30), heterogeneity in ligand binding can be resolved into an ensemble of 1:1 pseudo first-order reactions with a continuous distribution of binding constants.…”

mentioning

confidence: 99%

“…None of the injections led to a steady-state equilibrium during the injection phase. The classic approach for evaluating the SPR sensorgrams expressing the binding of proteins to surface-immobilized ligands is based on the assumption that complexes form with a 1:1 stoichiometry in a simple pseudo first-order reaction (27,28). The binding kinetics of this reaction relate the signal at time point t, the rate of association (k a ), the rate of dissociation (k d ), and the constant protein concentration (c MBL3 ) in the flow stream during the injection phase as: where S Max is the SPR signal at binding saturation, and t 0 is the time point at the start of injection.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The Role of Nanometer-Scaled Ligand Patterns in Polyvalent Binding by Large Mannan-Binding Lectin Oligomers

Gjelstrup

Kaspersen

Behrens

et al. 2012

The Journal of Immunology

View full text Add to dashboard Cite

Mannan-binding lectin (MBL) is an important protein of the innate immune system and protects the body against infection through opsonization and activation of the complement system on surfaces with an appropriate presentation of carbohydrate ligands. The quaternary structure of human MBL is built from oligomerization of structural units into polydisperse complexes typically with three to eight structural units, each containing three lectin domains. Insight into the connection between the structure and ligand-binding properties of these oligomers has been lacking. In this article, we present an analysis of the binding to neoglycoprotein-coated surfaces by size-fractionated human MBL oligomers studied with small-angle x-ray scattering and surface plasmon resonance spectroscopy. The MBL oligomers bound to these surfaces mainly in two modes, with dissociation constants in the micro to nanomolar order. The binding kinetics were markedly influenced by both the density of ligands and the number of ligand-binding domains in the oligomers. These findings demonstrated that the MBL-binding kinetics are critically dependent on structural characteristics on the nanometer scale, both with regard to the dimensions of the oligomer, as well as the ligand presentation on surfaces. Therefore, our work suggested that the surface binding of MBL involves recognition of patterns with dimensions on the order of 10–20 nm. The recent understanding that the surfaces of many microbes are organized with structural features on the nanometer scale suggests that these properties of MBL ligand recognition potentially constitute an important part of the pattern-recognition ability of these polyvalent oligomers.

show abstract

mentioning

confidence: 99%

mentioning

confidence: 99%

The Role of Nanometer-Scaled Ligand Patterns in Polyvalent Binding by Large Mannan-Binding Lectin Oligomers

Gjelstrup

Kaspersen

Behrens

et al. 2012

The Journal of Immunology

View full text Add to dashboard Cite

show abstract

“…The affinity constants of Fabs were assessed by surface plasmon resonance with the BIAcore 3000 instrument (Biacore AB, Uppsala, Sweden), according to the general procedure outlined by the manufacturer (8). The cysteine-rich domain of the Gal/GalNAc lectin heavy subunit, rLecA (11), kindly provided by W. A. Petri, Jr., University of Virginia, was immobilized onto a CM5 chip (Biacore) surface at a low density by amine coupling chemistry.…”

Section: Methodsmentioning

confidence: 99%

Improved Affinity of a Human Anti-Entamoeba histolyticaGal/GalNAc Lectin Fab Fragment by a Single Amino Acid Modification of the Light Chain

Tachibana

Matsumoto

Cheng

et al. 2004

Clin Vaccine Immunol

View full text Add to dashboard Cite

We previously produced, in Escherichia coli, a human monoclonal antibody Fab fragment, CP33, specific for the galactose-and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica. To prepare antibodies with a higher affinity to the lectin, recombination PCR was used to exchange Ser 91 Amebiasis caused by infection with the intestinal protozoan parasite Entamoeba histolytica is a notable parasitic disease in both developing and developed countries. It has been estimated that 50 million people develop amebic colitis and extraintestinal abscesses, resulting in up to 110,000 deaths annually (18). The development of immunoprophylaxis and accurate diagnostic tools is important for the control of amebiasis. The application of monoclonal antibodies is a promising avenue of research for improvement in diagnosis.We recently produced several human monoclonal antibody Fab fragments specific for E. histolytica in Escherichia coli by use of combinatorial immunoglobulin gene libraries constructed from the peripheral lymphocytes of a patient with an amebic liver abscess and from an asymptomatic cyst passer (1,14,17). One of the Fab clones, CP33, derived from the asymptomatic cyst passer, recognized the cysteine-rich domain of the heavy subunit of the galactose-and N-acetyl-D-galactosamineinhibitable (Gal/GalNAc) lectin (12) of E. histolytica (17). This clone exhibited neutralizing activities to amebic adherence and to erythrophagocytosis. Furthermore, we produced the Fab fragment fused with alkaline phosphatase for diagnostic purposes (16).Recombinant antibody technology makes it possible to introduce site-directed or random mutations in the original antibody gene (3-5, 13, 19). Residues in the complementaritydetermining region (CDR), especially in CDR3 of both the heavy and light chains of antibody, are considered responsible for high-energy interactions with antigen. Therefore, mutations at these residues will likely abolish antigen binding. However, an increased affinity may also occur by mutation if the native residue exhibits a negative effect on the interaction. In the Kabat numbering system, CDR3 of the light chain is the amino acid segment from position 89 to 97 (6, 20). The corresponding amino acid residues in CP33 were GlnGlnSerTyrSer ThrProArgThr (17). When an additional 13 light chains which constitute antilectin Fabs with the heavy chain of CP33 were analyzed, high variability was observed at positions 91, 92, 94, and 96 (17). As a first step in the affinity maturation of human antibodies to E. histolytica, we attempted to modify Fab clone CP33 by single-amino-acid substitutions of Ser 91 and Arg 96 in the light chain. MATERIALS AND METHODSSite-directed mutagenesis. Site-directed mutagenesis in the light chain gene of CP33 (17) was performed by recombination PCR (7). The plasmid vector pFab1-His2, containing the light and the Fd region of the heavy chain genes, was amplified by using two sets of primers, CP33L-S 91 X-For (5Ј-CAACTTACTAC TGTCAACAGNNNTACAGTAC-3Ј, where N is any nucleotide) and CP33L-...

show abstract

“…The most common is that the binding event is based on a different mechanism from that which the selected model describes, which, in turn, is often due to insufficient assay development. The second is due to the existence of local minima where the optimizer may be trapped (Karlsson and Larsson, 2004).…”

Section: Theorymentioning

confidence: 99%

“…The importance of using reference subtracted data, global fitting of the kinetic constants and handling of diffusion-limited interactions were discussed in the early era of real-time biosensor experiments (Karlsson and Fält, 1997;Morton and Myszka, 1998;Myszka et al, 1998). Recent developments in experimental design include double referencing and the use of multiple spots (Karlsson and Larsson, 2004;Rich and Myszka, 2000). For applications where throughput is more important than precision (e.g.…”

Section: Introductionmentioning

confidence: 99%

Kinetic determinations of molecular interactions using Biacore—minimum data requirements for efficient experimental design

Önell¹,

Andersson²

2005

J. Mol. Recognit.

View full text Add to dashboard Cite

Reliable kinetic estimates can be obtained from significantly less data than is commonly used today, particularly in the characterization of 1:1 interactions involving low molecular weight compounds and proteins. We have designed a rational and cost-effective strategy to determine kinetic constants using Biacore's surface plasmon resonance-based biosensors and show that the number of measurements necessary for accurate kinetic determinations can be greatly reduced, increasing sample throughput and saving sample material. Simulated and measured data for a range of possible 1:1 interactants were studied to find the minimum requirements of a data set for kinetic analysis. The results showed that kinetic constants in the region 10(4) < k(a) < 10(7) M(-1) s(-1) (association) and 10(-4) < k(d) < 10(-1) s(-1) (dissociation) could easily be determined in a 1:1 interaction model. Owing to the information-dense nature of Biacore data, only two sample concentrations were necessary to reliably determine the kinetics. A standard sample concentration series consisting of 10-fold dilutions between approximately 10 microM and approximately 1 nM consistently provided at least two concentrations with sufficient information about the interaction in this region. Determinations of the constants became increasingly unreliable outside this region. If the rate constants prove to be outside the specified region or the data fits poorly to the 1:1-MTL model, more experiments are required. General recommendations for the design of a cost-effective assay to deliver reliable kinetic measurements are provided.

show abstract

Affinity Measurement Using Surface Plasmon Resonance

Cited by 20 publications

References 5 publications

The Role of Nanometer-Scaled Ligand Patterns in Polyvalent Binding by Large Mannan-Binding Lectin Oligomers

The Role of Nanometer-Scaled Ligand Patterns in Polyvalent Binding by Large Mannan-Binding Lectin Oligomers

Improved Affinity of a Human Anti-Entamoeba histolyticaGal/GalNAc Lectin Fab Fragment by a Single Amino Acid Modification of the Light Chain

Kinetic determinations of molecular interactions using Biacore—minimum data requirements for efficient experimental design

Contact Info

Product

Resources

About