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1.
J Am Chem Soc ; 136(38): 13134-7, 2014 Sep 24.
Article in English | MEDLINE | ID: mdl-25190510

ABSTRACT

Stimulus-responsive hydrogel materials that stabilize and control protein dynamics have the potential to enable a range of applications that take advantage of the inherent specificity and catalytic efficiencies of proteins. Here we describe the modular construction of a hydrogel using an engineered calmodulin (CaM) within a poly(ethylene glycol) (PEG) matrix that involves the reversible tethering of proteins through an engineered CaM-binding sequence. For these measurements, maltose binding protein (MBP) was isotopically labeled with (13)C and (15)N, permitting dynamic structural measurements using TROSY-HSQC NMR spectroscopy. The protein dynamics is suppressed upon initial formation of hydrogels, with a concomitant increase in protein stability. Relaxation of the hydrogel matrix following transient heating results in enhanced protein dynamics and resolution of substrate-induced large-amplitude domain rearrangements.


Subject(s)
Calmodulin/chemistry , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Immobilized Proteins/chemistry , Maltose-Binding Proteins/chemistry , Polyethylene Glycols/chemistry , Skeletal Muscle Myosins/chemistry , Binding Sites , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Stability
2.
Anal Biochem ; 419(2): 333-5, 2011 Dec 15.
Article in English | MEDLINE | ID: mdl-21933659

ABSTRACT

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays can simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as with traditional ELISA diagnostics, endogenous antibodies in patient sera can cause interference. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit (i.e., a single-chain antibody fragment [scFv]) is an effective strategy for reducing assay interference. Our finding illustrates a source of error introduced by the reliance on immunoglobulin-based capture reagents in sandwich immunoassays with human serum samples. We demonstrate that scFvs can be used in such assays to improve reliability by reducing heterophilic antibody interference, thereby improving biomarker analysis and validation.


Subject(s)
Antibodies, Heterophile/immunology , Immunoassay/methods , Single-Chain Antibodies/immunology , Animals , Humans , Immunoglobulin G/blood , Mice , Single-Chain Antibodies/blood
3.
Biotechnol Bioeng ; 108(10): 2456-67, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21538339

ABSTRACT

A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, 3 also bound to full-length BoNT/A toxin complex with affinities ranging from 5 to 48 nM. Epitope binning showed that the three unique clones recognized at least two epitopes distinct from one another as well as from the detection MAbs. After production in E. coli, scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigens. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support.


Subject(s)
Antibodies, Bacterial/chemistry , Antibody Specificity , Botulinum Toxins, Type A/chemistry , Epitopes/chemistry , Single-Chain Antibodies/chemistry , Antibodies, Bacterial/biosynthesis , Antibodies, Bacterial/genetics , Botulinum Toxins, Type A/genetics , Botulinum Toxins, Type A/metabolism , Epitopes/genetics , Epitopes/metabolism , Humans , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Single-Chain Antibodies/biosynthesis , Single-Chain Antibodies/genetics
4.
Cancer Biomark ; 6(5-6): 271-9, 2010.
Article in English | MEDLINE | ID: mdl-20938087

ABSTRACT

Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (e.g., yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype--binding conferred by an antibody fragment--with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune libraries provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Biomarkers, Tumor/analysis , Neoplasms/diagnosis , Peptide Library , Proteomics/methods , Biomarkers, Tumor/immunology , Humans , Neoplasms/immunology
5.
Protein Eng Des Sel ; 22(5): 325-32, 2009 May.
Article in English | MEDLINE | ID: mdl-19321520

ABSTRACT

Antibodies are widely used for diagnostic and therapeutic applications because of their sensitive and specific recognition of a wide range of targets; however, their application is limited by their structural complexity. More demanding applications require greater stability than can be achieved by immunoglobulin-based reagents. Highly stable, protein-based affinity reagents are being investigated for this role with the goal of identifying a suitable scaffold that can attain specificity and sensitivity similar to that of antibodies while performing under conditions where antibodies fail. We have engineered Top7--a highly stable, computationally designed protein--to specifically bind human CD4 by inserting a peptide sequence derived from a CD4-specific antibody. Molecular dynamics simulations were used to evaluate the structural effect of the peptide insertion at a specific site within Top7 and suggest that this Top7 variant retains conformational stability over 100 degrees C. This engineered protein specifically binds CD4 and, consistent with simulations, is extremely resistant to thermal and chemical denaturation--retaining its secondary structure up to at least 95 degrees C and requiring 6 M guanidine to completely unfold. This CD4-specific protein demonstrates the functionality of Top7 as a viable scaffold for use as a general affinity reagent which could serve as a robust and inexpensive alternative to antibodies.


Subject(s)
Affinity Labels/chemical synthesis , Carrier Proteins/chemical synthesis , Computational Biology/methods , Models, Molecular , Protein Engineering/methods , Affinity Labels/metabolism , Amino Acid Sequence , CD4 Antigens/metabolism , Carrier Proteins/metabolism , Chromatography, Gel , Circular Dichroism , Computer Simulation , Enzyme-Linked Immunosorbent Assay , Humans , Mutagenesis , Sensitivity and Specificity
6.
Curr Protoc Cytom ; Chapter 4: Unit4.7, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18770649

ABSTRACT

These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks, resulting in a panel of scFvs specific for the target antigen.


Subject(s)
Antigens, Surface/immunology , Cloning, Molecular/methods , Immunoglobulin Fragments/metabolism , Peptide Library , Yeasts/immunology , Algorithms , Animals , Antibody Formation/genetics , Antibody Formation/physiology , Antigen-Antibody Reactions/immunology , Antigens, Surface/genetics , Antigens, Surface/metabolism , B-Lymphocytes/immunology , Cell Separation/methods , Humans , Immunoglobulin Fragments/genetics , Magnetics , Yeasts/genetics
7.
Proteomics ; 8(11): 2199-210, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18452230

ABSTRACT

Sandwich ELISA microarrays have great potential for validating disease biomarkers. Each ELISA relies on robust-affinity reagents that retain activity when immobilized on a solid surface or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional IgG. Unfortunately, scFv are typically less active than IgG following immobilization on a solid surface and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv constructs to determine a more robust strategy for using scFv as ELISA reagents. Two promising strategies emerged from these studies: (i) the precapture of epitope-tagged scFv using an antiepitope antibody and (ii) the direct printing of a thioredoxin (TRX)/scFv fusion protein on glass slides. Both strategies improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the antiepitope precapture method introduced a risk of reagent transfer. Using the direct printing method, we show that scFv against prostate-specific antigen (PSA) are highly specific when tested against 21 different IgG-based assays. In addition, the scFv microarray PSA assay gave comparable quantitative results (R(2) = 0.95) to a commercial 96-well ELISA when tested using human serum samples. In addition, we find that TRX-scFv fusions against epidermal growth factor and toxin X have good LOD. Overall, these results suggest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems.


Subject(s)
Antibodies/chemistry , Enzyme-Linked Immunosorbent Assay/instrumentation , Enzyme-Linked Immunosorbent Assay/methods , Proteomics/methods , Animals , Cell Separation , Epidermal Growth Factor/chemistry , Epitopes/chemistry , Humans , Immunoglobulin Fragments/chemistry , Immunoglobulin G/chemistry , Immunoglobulin Variable Region/chemistry , Mice , Protein Array Analysis/methods , Proteins/chemistry , Thioredoxins/chemistry
8.
Anal Biochem ; 371(1): 105-15, 2007 Dec 01.
Article in English | MEDLINE | ID: mdl-17718996

ABSTRACT

Antibody microarrays are an emerging technology that promises to be a powerful tool for the detection of disease biomarkers. The current technology for protein microarrays has been derived primarily from DNA microarrays and is not fully characterized for use with proteins. For example, there are a myriad of surface chemistries that are commercially available for antibody microarrays, but there are no rigorous studies that compare these different surfaces. Therefore, we have used a sandwich enzyme-linked immunosorbent assay (ELISA) microarray platform to analyze 17 different commercially available slide types. Full standard curves were generated for 23 different assays. We found that this approach provides a rigorous and quantitative system for comparing the different slide types based on spot size and morphology, slide noise, spot background, lower limit of detection, and reproducibility. These studies demonstrate that the properties of the slide surface affect the activity of immobilized antibodies and the quality of data produced. Although many slide types produce useful data, glass slides coated with aldehyde silane, poly-l-lysine, or aminosilane (with or without activation with a crosslinker) consistently produce superior results in the sandwich ELISA microarray analyses we performed.


Subject(s)
Antibodies/immunology , Oligonucleotide Array Sequence Analysis/instrumentation , Chemokine CCL5/immunology , E-Selectin/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Intercellular Adhesion Molecule-1/immunology , Male , Oligonucleotide Array Sequence Analysis/methods , Prostate-Specific Antigen/immunology , Reproducibility of Results , Sensitivity and Specificity , Surface Properties
9.
Front Biosci ; 12: 3956-64, 2007 May 01.
Article in English | MEDLINE | ID: mdl-17485349

ABSTRACT

Enzyme-linked immunosorbent assay (ELISA) microarrays promise to be a powerful tool for the detection of disease biomarkers. The original technology for printing ELISA microarray chips and capturing antibodies on slides was derived from the DNA microarray field. However, due to the need to maintain antibody structure and function when immobilized, surface chemistries used for DNA microarrays are not always appropriate for ELISA microarrays. In order to identify better surface chemistries for antibody capture, a number of commercial companies and academic research groups have developed new slide types that could improve antibody function in microarray applications. In this review we compare and contrast the commercially available slide chemistries, as well as highlight some promising recent advances in the field.


Subject(s)
Antibodies/immunology , Surface Properties , Enzyme-Linked Immunosorbent Assay
10.
Cell ; 114(1): 99-111, 2003 Jul 11.
Article in English | MEDLINE | ID: mdl-12859901

ABSTRACT

Phosphoinositides (PtdInsPs) play critical roles in cytoplasmic signal transduction pathways. However, their functions in the nucleus are unclear, as specific nuclear receptors for PtdInsPs have not been identified. Here, we show that ING2, a candidate tumor suppressor protein, is a nuclear PtdInsP receptor. ING2 contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. We find that the PHD fingers of ING2 and other diverse nuclear proteins bind in vitro to PtdInsPs, including the rare PtdInsP species, phosphatidylinositol 5-phosphate (PtdIns(5)P). Further, we demonstrate that the ING2 PHD finger interacts with PtdIns(5)P in vivo and provide evidence that this interaction regulates the ability of ING2 to activate p53 and p53-dependent apoptotic pathways. Together, our data identify the PHD finger as a phosphoinositide binding module and a nuclear PtdInsP receptor, and suggest that PHD-phosphoinositide interactions directly regulate nuclear responses to DNA damage.


Subject(s)
Apoptosis/genetics , Cell Nucleus/metabolism , DNA Damage/genetics , Eukaryotic Cells/metabolism , Homeodomain Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction/genetics , Tumor Suppressor Proteins , 1-Phosphatidylinositol 4-Kinase/metabolism , Amino Acid Sequence/genetics , Base Sequence/genetics , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Nucleus/genetics , Genes, Tumor Suppressor , Homeodomain Proteins/antagonists & inhibitors , Homeodomain Proteins/genetics , Humans , Molecular Sequence Data , Phosphatidylinositol Phosphates/metabolism , Protein Binding/genetics , Protein Structure, Tertiary/genetics , RNA Interference , Receptors, Cytoplasmic and Nuclear/genetics , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
11.
Anal Biochem ; 310(1): 93-9, 2002 Nov 01.
Article in English | MEDLINE | ID: mdl-12413478

ABSTRACT

Using Biacore's surface plasmon resonance-based biosensor technology, we developed experimental protocols and probed test conditions required to study drugs interacting with liposome surfaces. Liposome capture on hydrophobic alkane surfaces (Pioneer L1 chip) was reproducible and stable under variable conditions of pH, temperature, lipid content, cholesterol content, and buffer dimethylsulfoxide concentration. Importantly, drug binding responses were directly proportional to the amount of lipid captured, while the kinetics of drug binding and the magnitude of the responses correlated with a drug's chemical composition. In general, anionic drugs tended to rapidly dissociate from the surface, while cationic drugs displayed heterogeneous binding, suggesting partitioning within the lipid bilayer itself. The results illustrate how surface plasmon resonance can be used to establish passive transport properties of drugs.


Subject(s)
Liposomes/metabolism , Pharmaceutical Preparations/metabolism , Surface Plasmon Resonance/methods , Biosensing Techniques/methods , Cholesterol/chemistry , Dimethyl Sulfoxide/chemistry , Kinetics , Liposomes/chemistry , Membrane Fluidity , Pharmaceutical Preparations/chemistry , Phosphatidylcholines/chemistry , Reproducibility of Results , Surface Properties , Temperature
12.
Protein Sci ; 11(5): 1017-25, 2002 May.
Article in English | MEDLINE | ID: mdl-11967359

ABSTRACT

The binding interactions of small molecules with carbonic anhydrase II were used as model systems to compare the reaction constants determined from surface- and solution-based biophysical methods. Interaction data were collected for two arylsulfonamide compounds, 4-carboxybenzenesulfonamide (CBS) and 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), binding to the enzyme using surface plasmon resonance, isothermal titration calorimetry, and stopped-flow fluorescence. We demonstrate that when the surface plasmon resonance biosensor experiments are performed with care, the equilibrium, thermodynamic, and kinetic constants determined from this surface-based technique match those acquired in solution. These results validate the use of biosensor technology to collect reliable data on small molecules binding to immobilized macromolecular targets. Binding kinetics were shown to provide more detailed information about complex formation than equilibrium constants alone. For example, although carbonic anhydrase II bound DNSA with twofold higher affinity than CBS, kinetic analysis revealed that CBS had a fourfold slower dissociation rate. Analysis of the binding and transition state thermodynamics also revealed significant differences in the enthalpy and entropy of complex formation. The lack of labeling requirements, high information content, and high throughput of surface plasmon resonance biosensors will make this technology an important tool for characterizing the interactions of small molecules with enzymes and receptors.


Subject(s)
Carbonic Anhydrase II/chemistry , Dansyl Compounds/chemistry , Sulfonamides/chemistry , Animals , Cattle , Kinetics , Protein Binding , Substrate Specificity , Surface Plasmon Resonance , Thermodynamics
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