SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping.

Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides.

Epitope Mapping of Antibodies Using Bacterial Cell Surface Display of Gene Fragment Libraries.

The unique property of specific high affinity binding to more or less any target of interest has made antibodies tremendously useful in numerous applications. Hence, knowledge of the precise binding site (epitope) of antibodies on the target protein is one of the most important features for understanding its performance and determining its reliability in immunoassays. Here, we describe an updated protocol for high-resolution method for mapping epitopes of antibodies based on bacterial surface expression of antigen fragments followed by antibody-based flow cytometric analysis.

Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders.

Surface display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various display systems available, phage display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive bacterial host for display of a library of human antibody genes which, when combined with phage display, provides ease of use for screening, sorting and ranking by flow cytometry.

Stratification of responders towards eculizumab using a structural epitope mapping strategy.

The complement component 5 (C5)-binding antibody eculizumab is used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome (aHUS). As recently reported there is a need for a precise classification of eculizumab responsive patients to allow for a safe and cost-effective treatment. To allow for such stratification, knowledge of the precise binding site of the drug on its target is crucial.

Patient understanding of the importance of statin use in the perioperative period.

Objective: Perioperative hydroxy-3-methyl glutaryl coenzyme A reductase inhibitors (statins) have been shown to decrease morbidity and mortality after noncardiac surgery. The objective of this study was to assess patient understanding of the potential benefits of perioperative statins in a select population already on chronic therapy. A secondary aim was to determine the frequency with which patients recalled having a discussion with their provider regarding perioperative statins.

Proteome-wide epitope mapping of antibodies using ultra-dense peptide arrays.

Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis.

Epitope mapping of monoclonal and polyclonal antibodies using bacterial cell surface display.

The unique property of specific high-affinity binding to more or less any target of interest has made antibodies tremendously useful in numerous applications. Hence knowledge of the precise binding site (epitope) of antibodies on the target protein is one of the most important features for understanding its performance and determining its reliability in immunoassays. Here, we describe a high-resolution method for mapping epitopes of antibodies based on bacterial surface expression of antigen fragments followed by antibody-based flow cytometric sorting.

Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody.

One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity.

Renal safety of 1-hour pamidronate infusion for breast cancer and multiple myeloma patients: comparison between clinical trials and population-based database.

Purpose: The American Society of Clinical Oncology recommends infusion of pamidronate over 2 hours to avoid renal deterioration, although there are data to suggest that 1-hour infusions may be safe.

Methods: Prevalence of renal deterioration with 1-hour pamidronate infusions from a population database was compared to renal deterioration with 2-hour pamidronate infusions, in randomised, controlled, trials. A cost-minimisation analysis, comparing the 1- and 2-hour pamidronate infusions, and the 15-minute infusion of zoledronic acid, was performed with a sensitivity analysis that varied the opportunity cost of time in the treatment room.

Evaluation of predictive formulae for glomerular filtration rate for carboplatin dosing in gynecological malignancies.

Objectives: Carboplatin dosing is usually based on glomerular filtration rate (GFR). The Cockcroft-Gault and the Modified Diet in Renal Disease (MDRD) Study formulae are based on serum creatinine to estimate GFR when measured GFR is impractical. The MDRD formula has been shown to be more accurate in non-cancer patients with chronic renal disease.