Functional consequences of insertions and deletions in the complementarity-determining regions of human antibodies.
AuthorsLantto J, Ohlin M
JournalJ Biol Chem
PubMed ID12237318
'Insertions and deletions of nucleotides in the genes encoding the variable domains of antibodies are natural components of the hypermutation process, which may expand the available repertoire of hypervariable loop lengths and conformations. Although insertion of amino acids has also been utilized in antibody engineering, little is known about the ... More
Continuous-flow, on-line monitoring of biospecific interactions using electrospray mass spectrometry.
AuthorsHogenboom AC, de Boer AR, Derks RJ, Irth H
JournalAnal Chem
PubMed ID11534702
'A continuous-flow analytical screening system is presented using electrospray mass spectrometry to measure the interaction of biologically active compounds with soluble affinity proteins. The biochemical detection system is based on a solution-phase, homogeneous assay. In a first step, compounds to be screened (e.g., biotinylated compounds, concentration range 10-1,000 nmol/L) are ... More
'Array-based sensors provide an architecture for multianalyte sensing. In this paper, we report a new approach for array fabrication. Sensors are made by immobilizing different reactive chemistries on the surfaces of microspheres. Sensor arrays are prepared by randomly distributing a mixture of microsphere sensors on an optical substrate containing thousands ... More
Using affinity capillary electrophoresis to determine binding stoichiometries of protein-ligand interactions.
AuthorsChu YH, Lees WJ, Stassinopoulos A, Walsh CT
JournalBiochemistry
PubMed ID8075061
'We have developed a new method utilizing affinity capillary electrophoresis (ACE) for the determination of binding stoichiometries in biochemical systems. Using the same concentration of a ligand in the sample and the electrophoresis buffer, the appearance of an inverted peak corresponding to the free ligand in the resulting electropherogram provides ... More
Peptides, antibodies, and FRET on beads in flow cytometry: A model system using fluoresceinated and biotinylated beta-endorphin.
AuthorsBuranda T, Lopez GP, Keij J, Harris R, Sklar LA
JournalCytometry
PubMed ID10451503
'BACKGROUND: Particulate surfaces such as beads are routinely used as platforms for molecular assembly for fundamental and practical applications in flow cytometry. Molecular assembly is transduced as the direct analysis of fluorescence, or as a result of fluorescence resonance energy transfer. Binding of fluorescent ligands to beads sometimes alters their ... More
Determination of biotin in biotin-conjugated protein by measuring fluorescence polarization.
AuthorsShah D, Salbilla V, Richerson R, Brown W
JournalClin Chem
PubMed ID7955390
Determination of avidin and biotin by fluorescence polarization.
AuthorsSchray KJ, Artz PG, Hevey RC
JournalAnal Chem
PubMed ID3400873
Antibody-antigen binding constants determined in solution-phase with the threshold membrane-capture system: binding constants for anti-fluorescein, anti-saxitoxin, and anti-ricin antibodies.
AuthorsDill K, Lin M, Poteras C, Fraser C, Hafeman DG, Owicki JC, Olson JD
JournalAnal Biochem
PubMed ID8203727
Affinities of various monoclonal and polyclonal antibodies for fluorescein-containing antigens, saxitoxin and ricin, were determined by using a light addressable potentiometric sensor-based system (Threshold). The dissociation constants, determined from Scatchard plots, ranged from 2 x 10(-7) to approximately 3 x 10(-12) M. Dissociation constants for fluorescein and saxitoxin were compared ... More
Polyelectrolyte surface interface for single-molecule fluorescence studies of DNA polymerase.
AuthorsKartalov EP, Unger MA, Quake SR
JournalBiotechniques
PubMed ID12661156
We report the use of polyelectrolyte multilayers in a stable robust surface chemistry for specific anchoring of DNA to glass. The nonspecific binding of fluorescently tagged nucleotides is suppressed down to the single-molecule level, and DNA polymerase is active on the anchored DNA template. This surface-chemistry platform can be used ... More
Photoaffinity labeling of antibodies for applications in homogeneous fluoroimmunoassays.
AuthorsChang IN, Lin JN, Andrade JD, Herron JN
JournalAnal Chem
PubMed ID7762830
A homogeneous noncompetitive immunoassay based on photoaffinity labeling techniques is described. Using this method, a fluorophore (reporter) can be specifically attached to an antibody in the vicinity of its antigen-combining sites. Upon antigen binding, changes in the fluorescence spectrum of the reporter molecule are often observed. Two fluorophores, pyrene and ... More
Biotin-fluorophore conjugates with poly(ethylene glycol) spacers retain intense fluorescence after binding to avidin and streptavidin.
AuthorsGruber HJ, Marek M, Schindler H, Kaiser K
JournalBioconjug Chem
PubMed ID9258455
Conventional biotin-fluorophore conjugates with approximately 14 atom spacers lose most of their fluorescence when binding to avidin or streptavidin, as is demonstrated in the present study. This explains the unusual fact that only biotinylated marker enzymes, but not fluorescent biotins, are regularly used in bioanalytic assays. Novel biotin-spacer-fluorophore conjugates are ... More
Accurate titration of avidin and streptavidin with biotin-fluorophore conjugates in complex, colored biofluids.
AuthorsGruber HJ, Kada G, Marek M, Kaiser K
JournalBiochim Biophys Acta
PubMed ID9685643
A new fluorimetric assay is presented for the specific and reliable quantitation of >/=2 nM avidin and streptavidin. The assay is based on pronounced changes in the fluorescence properties of commercial fluorescein-biotin, or of a newly synthesized biotin-poly(ethylene glycol)-pyrene conjugate, which occur upon binding to avidin and streptavidin. Accurate measurement ... More
Protein micropatterns using a pH-responsive polymer and light.
AuthorsChristman KL, Maynard HD
JournalLangmuir
PubMed ID16114947
Protein and peptide microarrays are popular candidates for medical diagnostics because of the possibility for high sensitivity and simultaneous marker screening. To realize the potential of these arrays, new strategies for ligand patterning are needed. We report a method for patterning proteins that utilizes a pH-responsive polymer, deep ultraviolet (DUV) ... More
Direct-write fabrication of functional protein matrixes using a low-cost Q-switched laser.
AuthorsKaehr B, Ertas N, Nielson R, Allen R, Hill RT, Plenert M, Shear JB
JournalAnal Chem
PubMed ID16643014
We report the use of an inexpensive, small, and "turn-key" Q-switched 532-nm Nd:YAG laser as a source for nonlinear, direct-write protein microfabrication. In this approach, microJoule pulses (pulse widths, approximately 600 ps) are focused using high numerical aperture optics to submicrometer focal spots, creating instantaneous intensities great enough to promote ... More
Study of ligand-receptor interactions by fluorescence correlation spectroscopy with different fluorophores: evidence that the homopentameric 5-hydroxytryptamine type 3As receptor binds only one ligand.
AuthorsWohland T, Friedrich K, Hovius R, Vogel H
JournalBiochemistry
PubMed ID10393542
The 5-hydroxytryptamine receptor of type 3 was investigated by fluorescence correlation spectroscopy (FCS). Binding constants of fluorescently labeled ligands, the stoichiometry, and the mass of the receptor are readily accessible by this technique, while the duration of measurement is on the order of seconds to minutes. The receptor antagonist 1,2,3, ... More
Biotin-pyrene conjugates with poly(ethylene glycol) spacers are convenient fluorescent probes for avidin and streptavidin.
AuthorsMarek M, Kaiser K, Gruber HJ
JournalBioconjug Chem
PubMed ID9258456
Conventional biotin-fluorophore conjugates with approximately 14 atom spacers are strongly quenched when bound to avidin or streptavidin, whereas fluorescence becomes insensitive to receptor binding if typical fluorophores are linked to biotin via poly(ethylene glycol) (PEG) chains (Gruber et al., see the second of three papers in this issue). In the ... More
Detection of epitope-tagged proteins in flow cytometry: fluorescence resonance energy transfer-based assays on beads with femtomole resolution.
AuthorsBuranda T, Lopez GP, Simons P, Pastuszyn A, Sklar LA
JournalAnal Biochem
PubMed ID11700971
Epitope tagging of expressed proteins is a versatile tool for the detection and purification of the proteins. This approach has been used in protein-protein interaction studies, protein localization, and immunoprecipitation. Among the most popular tag systems is the FLAG epitope tag, which is recognized by three monoclonal antibodies M1, M2, ... More