Background: Measuring anti-spike protein antibodies in human plasma or serum is commonly used to determine prior exposure to SARS-CoV-2 infection and to assess the anti-viral protection capacity. According to the mass-action law, a lesser concentration of tightly binding antibody can produce the same quantity of antibody-antigen complexes as higher concentrations of lower affinity antibody. Thus, measurements of antibody levels reflect both affinity and concentration.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
April 2021
Every year, over 100 million units of donated blood undergo mandatory screening for HIV, hepatitis B, hepatitis C, and syphilis worldwide. Often, donated blood is also screened for human T cell leukemia-lymphoma virus, Chagas, dengue, Babesia, cytomegalovirus, malaria, and other infections. Several billion diagnostic tests are performed annually around the world to measure more than 400 biomarkers for cardiac, cancer, infectious, and other diseases.
View Article and Find Full Text PDFAcridinium 9-carboxylic acid derivatives have been extensively used as chemiluminescent labels in diagnostic assays. Triggering acridinium with basic hydrogen peroxide produces a highly strained dioxetanone intermediate, which converts into an acridone in an electronically excited state and emits light at 420-440 nm. Here, we introduce a novel acridinium-fluorescein construct emitting at 530 nm, in which fluorescein is covalently attached to the acridinium N-10 nitrogen via a propyl sulfonamide linker.
View Article and Find Full Text PDFMulticolor chemiluminescent acridinium derivatives were synthesized by attaching various common fluorophores to the N -acridinium position through a piperazine linker. Triggering of each acridinium derivative using alkaline hydrogen peroxide resulted in a chemiluminescence spectrum dominated by a strong emission (>95%) from the attached fluorophore. The highly quenched emission from the triggered acridinium, acting as a donor, points to a highly efficient intramolecular energy transfer in acridinium-based chemiluminophore-fluorophore tandems.
View Article and Find Full Text PDFBiotin-4-fluorescein (B4F) is a commonly used fluorescent probe for studying biotin-(strept)avidin interactions. During a characterization study of an anti-biotin antibody, using B4F as the probe, we noticed a discrepancy in the expected and experimentally determined number of biotin binding sites. Analytical testing showed that the biotin moiety in the probe undergoes a photosensitized oxidation to produce a mixture of biotin sulfoxides which has the potential to impact the quantitation of binding sites using this fluorescent probe.
View Article and Find Full Text PDFUnlike other known anti-fluorescein antibodies, the monoclonal antibody 43.1 is directed toward the fluorescein's carboxyl phenyl moiety. It demonstrates a very high affinity (KD ∼ 70 pM) and a fast association rate (kon ∼ 2 × 10(7) M(-1 ) s(-1) ).
View Article and Find Full Text PDFIn the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.
View Article and Find Full Text PDFWe describe a compact scanning confocal fluorescence microscope capable of detecting particles concentrations less than 100 particles∕ml in ~15 min. The system mechanically moves a cuvette containing ~3 ml of sample. A relatively large confocal volume is observed within the cuvette using a 1 mm pinhole in front of a detection PMT.
View Article and Find Full Text PDFThe molecular chaperone HSP90 has been shown to facilitate cancer cell survival by stabilizing key proteins responsible for a malignant phenotype. We report here the results of parallel fragment-based drug design approaches in the design of novel HSP90 inhibitors. Initial aminopyrimidine leads were elaborated using high-throughput organic synthesis to yield nanomolar inhibitors of the enzyme.
View Article and Find Full Text PDFTwo new proteins of approximately 70 amino acids in length, corresponding to an unnaturally-linked N- and C-helix of the ectodomain of the gp41 protein from the human immunodeficiency virus (HIV) type 1, were designed and characterized. A designed tripeptide links the C-terminus of the C-helix with the N-terminus of the N-helix in a circular permutation so that the C-helix precedes the N-helix in sequence. In addition to the artificial peptide linkage, the C-helix is truncated at its N-terminus to expose a region of the N-helix known as the "Trp-Trp-Ile" binding pocket.
View Article and Find Full Text PDFFluorescence methods are commonly used in pharmaceutical drug discovery to assay the binding of drug-like compounds to signaling proteins and other bio-particles. For binding studies of non-fluorescent compounds, a competitive format may be used in which the binding of the compound results in displacement of another fluorescently labeled ligand. Highly-sensitive measurements within nano-liter sized open probe volumes can be accomplished using a confocal epi-illumination geometry and thus key tools for such drug-binding studies include fluorescence correlation spectroscopy (FCS) and its related techniques.
View Article and Find Full Text PDFFluorescence correlation spectroscopy (FCS) enables direct observation of the translational diffusion of single fluorescent molecules in solution. When fluorescent hapten binds to antibody, analysis of FCS data yields the fractional amounts of free and bound hapten, allowing determination of the equilibrium binding constant. Equilibrium dissociation constants of anti-digoxin antibodies and corresponding fluorescein-labeled digoxigenin obtained by FCS and fluorescence polarization measurements are identical.
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