Fundamentals and Applications of Chromatographic Adsorption

The affinity and capacity of ion-exchange adsorbents of a given total-charge density are improved by immobilization of the charges in pre-ordered clusters, rather than individually in random locations. Here, we demonstrate that polyamine spermine can serve as an alternative to our previously described expensive peptide clustered ligands. Spermine Sepharose exhibited superior α-lactalbumin binding capacity (Qmax > 1.6 and 1.3-fold higher than those for Qiagen DEAE and GE DEAE Sepharose adsorbents of much greater charge density, respectively) and higher initial binding affinity (Qmax/KD 2.4 and 2.1-fold higher, respectively). The new spermine-based matrix also exhibited a higher value of the salt-sensitivity Z parameter, suggesting an increased number of apparent interaction sites between the protein and the resin, and functioned well in column mode. In another study, we developed an ultra-sensitive immunochromatographic lateral flow assay (LFA) for the detection of MS2 bacteriophage. LFAs are a well-established point-of-care diagnostic method, but the sensitivities of the standard gold and latex particle formats are not sufficient for many potential applications. Here we present a novel reporter, horseradish peroxidase (HRP)-labeled immuno-phage particles, for sensitivity-enhanced LFAs. Two different types of approaches were employed to manufacture antibody- and peroxidase-doubly-modified M13 bacteriophage, and these novel reporters were successfully integrated into an LFA. The sensitivity of the assay for a model viral pathogen, MS2, was 1000-fold greater than the traditional gold nanoparticle LFA with the same antibodies and results could easily be visually assessed, even without instruments. In another study in collaboration with the group of C. Landes of Rice University, we employed a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT) to study complex mechanistic details involved in protein adsorption. We demonstrated that clusters of charges are necessary to create detectable adsorption sites and that even chemically-identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Simulated elution profiles predicted from the molecular-scale data suggest that if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five.Biology and Biochemistry, Department o

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally

Aptamer-Phage Reporters for Ultrasensitive Lateral Flow Assays

We introduce the modification of bacteriophage particles with aptamers for use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an in vivo biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin朾iotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from Staphylococcus aureus (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ?100 times lower than those of previously reported IgE assays

Detection of Viruses By Counting Single Fluorescent Genetically Biotinylated Reporter Immunophage Using a Lateral Flow Assay

We demonstrated a lateral flow immunoassay (LFA) for detection of viruses using fluorescently labeled M13 bacteriophage as reporters and single-reporter counting as the readout. AviTag-biotinylated M13 phage were functionalized with antibodies using avidin–biotin conjugation and fluorescently labeled with AlexaFluor 555. Individual phage bound to target viruses (here MS2 as a model) captured on an LFA membrane strip were imaged using epi-fluorescence microscopy. Using automated image processing, we counted the number of bound phage in micrographs as a function of target concentration. The resultant assay was more sensitive than enzyme-linked immunosorbent assays and traditional colloidal-gold nanoparticle LFAs for direct detection of viruses

Sensitive Detection of Norovirus Using Phage Nanoparticle Reporters in Lateral-Flow Assay

<div><p>Noroviruses are recognized worldwide as the principal cause of acute, non-bacterial gastroenteritis, resulting in 19-21 million cases of disease every year in the United States. Noroviruses have a very low infectious dose, a short incubation period, high resistance to traditional disinfection techniques and multiple modes of transmission, making early, point-of-care detection essential for controlling the spread of the disease. The traditional diagnostic tools, electron microscopy, RT-PCR and ELISA require sophisticated and expensive instrumentation, and are considered too laborious and slow to be useful during severe outbreaks. In this paper we describe the development of a new, rapid and sensitive lateral-flow assay using labeled phage particles for the detection of the prototypical norovirus GI.1 (Norwalk), with a limit of detection of 10⁷ virus-like particles per mL, one hundred-fold lower than a conventional gold nanoparticle lateral-flow assay using the same antibody pair.</p></div

Aptamer-Phage Reporters for Ultrasensitive Lateral Flow Assays

We introduce the modification of bacteriophage particles with aptamers for use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an <i>in vivo</i> biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin–biotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from <i>Staphylococcus aureus</i> (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ∼100 times lower than those of previously reported IgE assays

Detection of Norwalk VLPs using a sandwich ELISA.

<p>A) Antibody pair screening for the detection of Norwalk VLPs; values correspond to the absorbance for a sample for 10⁹ VLPs offered; background absorbance for no VLP sample was subtracted (typical value ~0.1). Red color denotes maximum ΔOD450 observed in the ELISA, yellow lowest, and a smooth color gradient in between. Black box denotes the sandwich pair that was used in LFA. B) Sandwich ELISA detecting Norwalk VLPs where F2 was used as the capturing antibody. For the detection biotinylated F1 and streptavidin HRP (antibody sandwich, closed symbols), or the phage construct (Antibody-NeutrAvidin-AviTag phage) and anti-M13/ HRP conjugate (phage sandwich; open symbols) were used.</p