Methodologies for the isolation of alternative binders with improved clinical potentiality over conventional antibodies

The availability of binders to different functional domains of the same protein or to physiologically co-operating proteins allows for the simultaneous inhibition of independent downstream signaling pathways. This multi-target approach represents a promising therapeutic strategy, as demonstrated in the case of the synergistic effect of anti-Her2 treatment based on the combined use of the trastuzumab and pertuzumab monoclonal antibodies that induce cellular cytotoxicity and impair the receptor dimerization, respectively. Therefore, a reliable selection method for the recovery of epitope-specific antibodies is highly needed. Animal immunization with short peptides resembling the epitope sequence for raising conventional antibodies represents an alternative. Panning phage displayed libraries of recombinant antibodies such as scFvs and nanobodies or of other peptide collections is another option. Although recombinant antibodies can provide the same specificity as conventional antibodies, they offer at least two further advantages: i) the protocols for the selection of epitope-specific antibodies can be rationally designed, and ii) their expression as multivalent, bispecific and biparatopic molecules is feasible. This review will analyze the recent literature concerning technical aspects related to the isolation, the expression as multivalent molecules, and the therapeutic applications of binders able to interfere with antigen functional domains. The term binder will be preferred when possible to include those molecules, such as peptides or affibodies, with at least some proven practical uses.     

A method for efficient gene isolation from phage lambda gt11 libraries: use of antisera to denatured, acetone-precipitated proteins

Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins.     

Recovery of proteins on a milligram scale from polyacrylamide electrophoresis gels,exemplified by purification of a retinol-binding protein

An apparatus suitable for the recovery of proteins from polyacrylamide gels on a milligram scale by displacement electrophoresis (isotachophoresis) is described along with a buffer system that is suitable for this purpose with most proteins. The technique is illustrated by the recovery of a protein from a 15% polyacrylamide gel. The recovery was almost quantitative and the eluted protein showed little contamination upon quantitative amino acid analysis and automatic Edman degradation.     

Electrophoretic elution of nucleic acids from acrylamide and agarose gels

A simple method for electrophoretic elution of nucleic acids from gel slices is described. The procedure utilizes a standard tube gel system and can be completed in as little as one hour. Nucleic acids are recovered in a small volume with almost 100% efficiency. The procedure is applicable equally to acrylamide and agarose gels, and small as well as large RNA and DNA molecules. The eluted nucleic acids are essentially undegraded and are suitable for a variety of structural and biological analyses.     

Elution of low molecular weight solutes from viable cells of Saccharomyces bisporus

Previous work has shown that high molecular weight compounds were released from Saccharomyces bisporus by -mercaptoethanol, 2 M KCl, 0.5 M KCl and osmotic shock without affecting viability of the cells. In this current experiment, it was shown that low molecular weight compounds were also eluted when cells were treated in sequence with the same reagents. Alanine, glutamate, serine, an unidentified amino acid, glucose, glycerol, and arabitol were all eluted by each of the first three reagents. The osmotic shock eluate contained a larger number and quantity of amino acids than the first three eluates but, otherwise, the compounds in this eluate were the same. One hundred percent of the cellular glycerol and 65–70% of the total amounts of the other above mentioned solutes were released by the 4 eluting treatments. A hot water treatment was needed to extract the remainder of these solutes. The hot water extract also contained almost all the cellular proline. It was suggested that the elutable solutes are contained by cells in compartments (or vesicles) whose membranes are accessible to the eluting reagents without affecting the plasmalemma.     

Investigation of the enantioselectivity and enantiomeric elution order of some piperidine-2,6-dione drugs on chiralcel OD column

The enantioselectivity and enantiomeric separation of five racemic piperidine-2,6-dione compounds, on the cellulose tris(3,5-dimethylphenyl carbamate) chiral stationary phase Chiralcel OD-CSP were investigated under the same chromatographic conditions. This class of drugs includes glutethimide, aminoglutethimide, cyclohexylaminoglutethimide, pyridoglutethimide, and phenglutarimide. The results revealed that chiral recognition and the binding sites of these drugs on the Chiralcel OD column are similar, regardless of the absolute configuration of the individual enantiomers. A possible chiral recognition mechanism(s) for this class of drugs and the CSP is presented. © 1994 Wiley-Liss, Inc.     

Circular dichroism detection in HPLC

A circular dichroism-based detection system presents several advantages in the HPLC analysis of chiral compounds because of the selective monitoring of optically active molecules. Its use allows reliable determination of enantiomeric excesses and elution order. To this end, the application of empirical, semiempirical, and nonempirical methods to get stereochemical information from the CD signal is reported. Furthermore, recording the CD spectra on line and evaluation of the dissymetry factor make the CD detection very powerful in characterizing the stereochemistry of chiral eluates.     

The molecular size of glycans liberated by hydrazinolysis from semliki forest virus proteins

The glycans of well characterized, [6-³H]galactose-labelled glycopeptides, GC-4 from bovine IgG₁ as well as GP-V-2 and GP-V-5 from α₁-acid glycoprotein, were liberated by hydrazinolysis. Molecular weights close to the expected values were observed by gel filtration. Desialated glycans of Semliki Forest virus proteins were likewise liberated by hydrazinolysis and subjected to gel filtration. Metabolically labelled [1-³H]galactose-oligosaccharides of the mixed viral proteins revealed an apparent molecular weight of 1800. The bi-antennary glycan liberated from the reference glycopeptide GC-4 was of 1750 daltons. A mixture of [2-³H]mannose-labelled E₁-and E₂-proteins of the virus contained L-type glycans of 1800 daltons (formerly called A-type), and M-type glycans of 1200 daltons (formerly called B-type). A fraction of the E₃-glycans isolated by affinity chromatography on Concanavalin A-Sepharose showed an average molecular weight of 2150, a value intermediate between the three- and four-antennary glycans liberated from the reference glycopeptides GP-V-5 and GP-V-2. The rest of the E₃-glycans were of 1850 daltons, a value close to the bi-antennary GC-4 glycan. We suggest that the comparatively large size of the E₃-glycans and the exposed position of E₃-proteins on the viral surface may be interrelated.     

Development and validation of an experimental and theoretical method for the chiral discrimination of dinotefuran

Dinotefuran is a low-cost agrochemical considered a highly toxic product. In this sense, there is a need for its constant environmental, biological, and food control, aiming to ensure its use to humans as well as to preserve biodiversity and ecosystems. In the present work, we developed an experimental and theoretical method for dinotefuran chiral discrimination. According to the main results, the dinotefuran enantioselective separation was efficiently optimized by high-performance liquid chromatography evaluating the influence of different percentage compositions in the mobile phase to improve the resolution of the peaks in the chromatogram. The novelty of this work was the proposition of a reduced molecular model for the chiral selector amylose-Tris-(3,5-dimethylphenylcarbamate) polysaccharide that was able to adequately describe at the molecular level its interaction with the dinotefuran enantiomers. Besides, the thermodynamic and structural parameters obtained via density functional theory calculations pointed out the chiral discrimination as well as the enantiomeric elution order of the analyte studied, confirming the experimental data, thus validating our proposed method. Finally, hydrogen bonds and repulsive interactions played a key role in the discrimination between the diastereomeric complexes, and consequently, for the dinotefuran enantioselective separation.     

Evaluation of affinity microcolumns containing human serum albumin for rapid analysis of drug–protein binding

This study examined the use of affinity microcolumns as tools for the rapid analysis and high-throughput screening of drug–protein binding. The protein used was immobilized human serum albumin (HSA) and the model analytes were warfarin and l-tryptophan, two solutes often used as site-specific probes for drug binding to Sudlow sites I and II of HSA, respectively. The use of HSA microcolumns in binding studies was examined by using both zonal elution and frontal analysis formats. The zonal elution studies were conducted by injecting the probe compounds onto HSA microcolumns of varying lengths while measuring the resulting retention factors, plate heights and peak asymmetries. A decrease in the retention factor was noted when moving from longer to shorter column lengths while using a constant amount of injected solute. However, this change could be corrected, in part, by determining the relative retention factor of a solute versus a reference compound injected onto the same microcolumn. The plate height values were relatively consistent for all column lengths and gave an expected increase at higher linear velocities. The peak asymmetries were similar for all columns up to 1 mL/min but shifted to larger values at higher flow rates and when using short microcolumns (e.g., 1 mm length). The association equilibrium constants and number of binding sites estimated by frontal analysis for warfarin with HSA were consistent at the various column sizes that were tested and gave good agreement with previous literature values. These results confirmed affinity microcolumns provide comparable results to those obtained with longer columns and can be used in the rapid analysis of drug–protein binding and in the high-throughput screening of such interactions.