Isolation and functional analysis of phage-displayed antibody fragments targeting the staphylococcal superantigen-like proteins.

Staphylococcus aureus produces numerous virulence factors that manipulate the immune system, helping the bacteria avoid phagocytosis. In this study, we are investigating three immune evasion molecules called the staphylococcal superantigen-like proteins 1, 5, and 10 (SSL1, SSL5, and SSL10). All three SSLs inhibit vital host immune processes and contribute to S.

Isolation and characterization of phage display-derived scFv antibodies against human parechovirus 1 VP0 protein.

Human parechoviruses (PeVs) are common viruses that are associated with a variety of diseases from mild gastrointestinal and respiratory symptoms to severe central nervous system infections. Until now there has not been antibodies for visualizing parechovirus infection. We used E.

Combinatorial mutagenesis with alternative CDR-L1 and -H2 loop lengths contributes to affinity maturation of antibodies.

Loop length variation in the complementary determining regions (CDRs) 1 and 2 encoded in germline variable antibody genes provides structural diversity in naïve antibody libraries. In synthetic single framework libraries the parental CDR-1 and CDR-2 length is typically unchanged and alternative lengths are provided only at CDR-3 sites. Based on an analysis of the germline repertoire and structure-solved anti-hapten and anti-peptide antibodies, we introduced combinatorial diversity with alternative loop lengths into the CDR-L1, CDR-L3 and CDR-H2 loops of anti-digoxigenin and anti-microcystin-LR single chain Fv fragments (scFvs) sharing human IGKV3-20/IGHV3-23 frameworks.

Recombinant Antibodies with Unique Specificities Allow for Sensitive and Specific Detection of Uncarboxylated Osteocalcin in Human Circulation.

Osteocalcin is a bone-specific protein which contains three glutamic acid residues (Glu) that undergo post-translational gamma-carboxylation. Uncarboxylated osteocalcin (ucOC) may participate in the regulation of glucose metabolism, thus measurement of ucOC could be useful in evaluating interactions between bone and glucose metabolism. We developed recombinant antibodies and immunoassay to specifically detect ucOC in human blood samples.

Identification and analysis of anti-HDL scFv-antibodies obtained from phage display based synthetic antibody library.

Objective: In epidemiological studies plasma high density lipoprotein cholesterol (HDL-C) levels are found to correlate inversely with atherosclerotic cardiovascular events. HDL consists of different subpopulations and they vary in their anti-atherogenic properties. The aim of this study is to isolate coronary artery disease (CAD) specific anti-HDL scFv-antibodies.

Phage display aided improvement of a unique prostate-specific antigen (PSA) antibody unreactive with Lys(145)-Lys(146) internally cleaved forms.

Prostate specific antigen (PSA) is a commonly used marker of prostate cancer. A panel of four kallikrein immunoassays has been reported to improve the prediction of prostate biopsy outcome (cancer vs benign) in men with elevated PSA in the circulation. Assay of one of the kallikrein forms, intact free PSA (fPSA-I), is based on a unique monoclonal antibody (4D4), which is specific for PSA without the internal cleavage at Lys(145)-Lys(146).

Chimeric recombinant antibody fragments in cardiac troponin I immunoassay.

Objectives: To introduce a novel nanoparticle-based immunoassay for cardiac troponin I (cTnI) utilizing chimeric antibody fragments and to demonstrate that removal of antibody Fc-part and antibody chimerization decrease matrix related interferences.

Design And Methods: A sandwich-type immunoassay for cTnI based on recombinant chimeric (mouse variable/human constant) antigen binding (cFab) antibodies and intrinsically fluorescent nanoparticles was developed. To test whether using chimeric antibody fragments helps to avoid matrix related interferences, samples (n=39) with known amounts of triglycerides, bilirubin, rheumatoid factor (RF) or human anti-mouse antibodies (HAMAs) were measured with the novel assay, along with a previously published nanoparticle-based research assay with the same antibody epitopes.

Lactobacillus helveticus MIMLh5-specific antibodies for detection of S-layer protein in Grana Padano protected-designation-of-origin cheese.

Single-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein of Lactobacillus helveticus MIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein of L.

Two ScFv antibody libraries derived from identical VL-VH framework with different binding site designs display distinct binding profiles.

In directed evolution experiments, a single randomization scheme of an antibody gene does not provide optimal diversity for recognition of all sizes of antigens. In this study, we have expanded the recognition potential of our universal library, termed ScFvP, with a second distinct diversification scheme. In the second library, termed ScFvM, diversity was designed closer to the center of the antigen binding site in the same antibody framework as earlier.

Selection of stable scFv antibodies by phage display.

ScFv fragments are popular recombinant antibody formats but often suffer from limited stability. Phage display is a powerful tool in antibody engineering and applicable also for stability selection. ScFv variants with improved stability can be selected from large randomly mutated phage displayed libraries with a specific antigen after the unstable variants have been inactivated by heat or GdmCl.

Primer extension mutagenesis powered by selective rolling circle amplification.

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A.

Use of high-capacity surface with oriented recombinant antibody fragments in a 5-min immunoassay for thyroid-stimulating hormone.

High-capacity surfaces can enhance analyte-binding kinetics and be beneficial for rapid immunoassays. Site-specifically immobilized, oriented recombinant single-chain Fv (scFv) and Fab antibody fragments were compared with a conventional, nonoriented monoclonal antibody (Mab) to capture antigen from serum to solid surface in a one-step, two-site thyroid-stimulating hormone (TSH) immunoassay with a 5-min incubation time. The assay used a ready-to-use dry reagent-based concept and time-resolved fluorescent measurement.

Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage.

Phage display is a commonly used selection technique in protein engineering, but not all proteins can be expressed on phage. Here, we describe the expression of a cytoplasmic homodimeric enzyme dihydropteroate synthetase (DHPS) on M13 phage, established by protein engineering of DHPS. The strategy included replacement of cysteine residues and screening for periplasmic expression followed by random mutagenesis and phage display selection with a conformation-specific anti-DHPS antibody.

Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions.

Stability of single-chain Fvs (scFvs) can be improved by mutagenesis followed by phage display selection where the unstable variants are first inactivated by, for example, denaturing treatment. Here we describe a modified strategy for the selection of stabilized antibody fragments by phage display, based on denaturation under reducing conditions. This strategy was applied to an anti-thyroid-stimulating hormone (TSH) scFv fragment which refolded remarkably during the selection if denaturation was carried out in conventionally used non-reducing conditions.

Generic lanthanide fluoroimmunoassay for the simultaneous screening of 18 sulfonamides using an engineered antibody.

Sulfa antibiotics (sulfonamides) are used in veterinary and human medicine for therapeutic and prophylactic purposes. Veterinary use can result in foodstuffs derived from animals being contaminated with residual sulfonamides. Current sulfonamide-screening methods (mainly based on bacterial growth inhibition) are slow and inaccurate, since sensitivities of bacteria to different sulfonamides vary a lot.

Engineering of a broad specificity antibody for simultaneous detection of 13 sulfonamides at the maximum residue level.

Sulfa antibiotics (sulfonamides) are a group of molecules sharing the p-aminobenzenesulfonamide moiety. Sulfonamides are used in veterinary and human medicine. Sometimes, the meat or milk of medicated animals is contaminated with residual sulfonamides.