ABSTRACT
Microarray-based binding assays facilitate the discovery of protein ligands from large collections of small molecules. Hundreds of ligands can be identified, yet only a small portion of them have interfering effects (competitive or noncompetitive) on a specific protein-receptor binding reaction. Further efficient screening of ligands for those with specific modifying effect is needed in order to take the full advantage of throughputs of microarray-based assays for drug discovery. We report a label-free "microarray-in-microplate" assay platform for simultaneous acquisition of at least 32 dose-response curves in a single experiment, each curve having 12 concentration points. When combined with ligand discovery, this makes the microarray-based platform a true high-throughout means of finding inhibitors to specific protein-receptor reactions starting from a large collection of small-molecule libraries.
Subject(s)
Biological Assay/methods , Dose-Response Relationship, Drug , Microarray Analysis/instrumentation , Immobilized Proteins , Ligands , Staining and LabelingABSTRACT
Monoclonal antibodies (mAbs) are major reagents for research and clinical diagnosis. For their inherently high specificities to intended antigen targets and thus low toxicity in general, they are pursued as one of the major classes of new drugs. Yet binding properties of most monoclonal antibodies are not well characterized in terms of affinity constants and how they vary with presentations and/or conformational isomers of antigens, buffer compositions, and temperature. We here report a microarray-based label-free assay platform for high-throughput measurements of monoclonal antibody affinity constants to antigens immobilized on solid surfaces. Using this platform we measured affinity constants of over 1410 rabbit monoclonal antibodies and 46 mouse monoclonal antibodies to peptide targets that are immobilized through a terminal cysteine residue to a glass surface. The experimentally measured affinity constants vary from 10 pM to 200 pM with the median value at 66 pM. We compare the results obtained from the microarray-based platform with those from a benchmarking surface-plasmon-resonance-based (SPR) sensor (Biacore 3000).
Subject(s)
Antibodies, Monoclonal/immunology , Antibody Affinity/immunology , Antigen-Antibody Reactions/immunology , Animals , Antigens/immunology , High-Throughput Screening Assays/methods , Mice , Protein Array Analysis/methods , Rabbits , Surface Plasmon ResonanceABSTRACT
We explored two macromolecular scaffolds, bovine serum albumin (BSA) and polyvinyl alcohol (PVA), as chemically complementary platforms for immobilizing small molecule compounds on functionalized glass slides. We conjugated biotin molecules to BSA and amine-derivatized PVA and subsequently immobilized the conjugates on epoxy-functionalized glass slides through reaction of free amine residues on BSA and PVA with surface-bound epoxy groups. We studied binding reactions of such immobilized small molecule targets with solution-phase protein probes using an oblique-incidence reflectivity difference scanning optical microscope. The results showed that both BSA and amine-derivatized PVA were effective and efficient as carriers of small molecules with NHS residues and fluoric residues and for immobilization on epoxy-coated solid surfaces. A significant fraction of the conjugated small molecules retain their innate chemical activity.
Subject(s)
Ligands , Protein Array Analysis/methods , Serum Albumin, Bovine/chemistry , Animals , Biotin/chemistry , Biotin/immunology , Cattle , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , Kinetics , Polyvinyl Alcohol/chemistry , Protein BindingABSTRACT
We studied the effect of fluorescently labeling proteins on protein-ligand reactions. Unlabeled ligands (streptavidin-binding peptides and rabbit immunoglobulin G (IgG) as antigen targets) are immobilized on epoxy-functionalized glass slides. Unlabeled and Cy3-labeled protein probes from the same batch (streptavidin and goat antibodies) subsequently react with the surface-immobilized targets. By monitoring in situ the surface mass density change using an oblique-incidence reflectivity difference scanning microscope (a label-free detector), we measured k(on) and k(off) for streptavidin-peptide reactions and antibody-antigen reaction. We found that (1) equilibrium dissociation constants, defined as K(D) = k(off)/k(on), for streptavidin-peptide reactions increases by a factor of 3-4 when the solution-phase streptavidin is labeled with Cy3 dye and (2) K(D) for reactions of solution-phase goat anti-rabbit antibodies with rabbit IgG targets also change significantly when the goat antibodies are labeled with Cy3 dye.
Subject(s)
Carbocyanines/chemistry , Immunoglobulin G/chemistry , Oligopeptides/chemistry , Streptavidin/chemistry , Animals , Epoxy Compounds/chemistry , Glass/chemistry , Kinetics , Ligands , Rabbits , Surface Properties , Time FactorsABSTRACT
We applied oblique-incidence reflectivity difference microscopes (a form of polarization-modulated nulling ellipsometry) to detection of biomolecular microarrays without external labeling in a study of protein reactions with surface-immobilized targets. We show that the optical reflectivity difference signals can be quantitatively related to changes in surface mass density of molecular layers as a result of the reactions. Our experimental results demonstrate the feasibility of using oblique-incidence reflectivity difference microscopes for high-throughput proteomics research such as screening unlabeled protein probes against libraries of surface-immobilized small molecules.
Subject(s)
Immunoglobulin G/metabolism , Serum Albumin, Bovine/metabolism , Animals , Cattle , Humans , Microscopy/instrumentation , Microscopy/methods , Protein Array Analysis/methods , Protein Binding , Surface PropertiesABSTRACT
We describe a novel scanning optical microscope based on a polarization-modulated nulling ellipsometry. The new microscope employs a combination of scanning mirror and sample translation and thus enables high-throughput label-free detection of biomolecular microarrays with more than 10 000 protein or small-molecule targets. For illustration, we show the image of a 2760-spot protein microarray on a functionalized glass slide obtained with such a microscope. The new scanning microscope is also capable of determining, in parallel, the real-time binding kinetics of multiple molecular species under aqueous conditions.
Subject(s)
Image Enhancement/instrumentation , Microarray Analysis/instrumentation , Microscopy, Confocal/instrumentation , Robotics/instrumentation , Equipment Design , Equipment Failure Analysis , Image Enhancement/methods , Microarray Analysis/methods , Microscopy, Confocal/methods , Reproducibility of Results , Robotics/methods , Sensitivity and Specificity , Staining and LabelingABSTRACT
We show that reflection of a monochromatic light from a semi-infinite medium covered with a stack of layered media is equivalent to that from an effective "semi-infinite medium" characterized by two distinctive optical dielectric constants for the s-polarized and p-polarized components, respectively. Such an effective-substrate approach simplifies the analysis of ellipsometry measurements of a wide range of surface-bound processes including thin film growth and surface-bound reactions.
ABSTRACT
We studied the incidence-angle dependence of the optical reflectivity difference in response to ultrathin films on transparent and opaque substrates. We found that the classical three-layer model reproduces the experimentally obtained angular dependence for a monolayer of xenon on Nb(110) and for a monolayer of protein molecules on functionalized glass. We explore the enhancement of the optical response near the Brewster angle (or its equivalent for opaque substrates) in thin film detection.
Subject(s)
Algorithms , Image Interpretation, Computer-Assisted/methods , Membranes, Artificial , Models, Chemical , Refractometry/methods , Serum Albumin, Bovine/analysis , Serum Albumin, Bovine/chemistry , Adsorption , Coated Materials, Biocompatible/analysis , Coated Materials, Biocompatible/chemistry , Computer Simulation , Protein Binding , Scattering, RadiationABSTRACT
We developed an oblique-incidence reflectivity difference (OI-RD) scanning microscope for label-free imaging of microarrays of biomolecules upon solid substrates. We demonstrate that hybridization reactions in an oligonucleotide microarray fabricated upon a glass slide can be detected by such an OI-RD microscope.
Subject(s)
Biochemistry/methods , Microscopy/methods , Models, Theoretical , Oligonucleotide Array Sequence Analysis , Biochemistry/instrumentation , Equipment Design , Microscopy/instrumentation , Nucleic Acid Hybridization , Scattering, RadiationABSTRACT
Pharmacotherapeutic agents are uncommonly associated with hiccups. Corticosteroids and benzodiazepines have been the drug classes mentioned most frequently in the literature as being associated with the development of hiccups. However, by using a strict criterion, there is currently insufficient evidence for any drug to be considered causative in the etiology of hiccups.
Subject(s)
Drug-Related Side Effects and Adverse Reactions , Hiccup/chemically induced , HumansABSTRACT
To date, the stated program objectives have been met. There is a heightened awareness of ADRs, and the program has had a positive impact on patient care. More work is needed in the prevention of ADRs as opposed to their tabulation. Future educational efforts will focus on how reporting suspected ADRs can positively impact patient care.