Phosphotyrosyl peptides and analogues as substrates and inhibitors of purple acid phosphatases.

Purple acid phosphatases are metal-containing hydrolases. While their precise biological role(s) is unknown, the mammalian enzyme has been linked in a variety of biological circumstances (e.g.

The moral case for the legalisation of voluntary euthanasia.

If a person is suffering from illness or disability and wishes to end their life the law ought to facilitate rather than frustrate that choice argues Graham Oddie in this article. He points out the inconsistencies in current medical practice, and the gross disparity between the practice and the letter of the law. In dismissing many of the commonly raised objections to calls for reform of the law permitting euthanasia he makes a strong case for consistency in our approach to the right to die and patient autonomy.

Single-chain Fv of anti-idiotype 11-1G10 antibody interacts with antibody NC41 single-chain Fv with a higher affinity than the affinity for the interaction of the parent Fab fragments.

A single-chain Fv (scFv) fragment of anti-idiotype antibody 11-1G10, which recognizes an idiotope of anti-neuraminidase antibody NC41, was constructed by joining VH and VL domains with a (Gly4Ser)3 linker, with a pelB leader sequence, and two C-terminal FLAG tag sequences, and expressed in E. coli (10 mg/L). The 11-1G10 scFv was isolated by affinity chromatography on an anti-FLAG M2 antibody column as a 2:1 mixture of monomer and dimer forms which were separated by Superdex 75 chromatography; monomer (at 100 microg/ml) was stable for 7 days at 21 degrees C and 30 days at 4 degrees C, whereas the dimer slowly dissociated to monomer to yield a 2:1 monomerdimer equilibrium mixture after 30 days at 4 degrees C.

Nonspecific amine immobilization of ligand can Be a potential source of error in BIAcore binding experiments and may reduce binding affinities.

The interaction of monovalent forms of NC41, an anti-viral neuraminidase antibody, and the antiidiotype antibody 11-1G10 has been used as a model system for BIAcore analysis to demonstrate the potential problems resulting from the nonspecific amine coupling procedure. To avoid complications due to antibody bivalency, monovalent Fab fragments and monomeric recombinant scFvs were used. When immobilized by amine coupling, the 11-1G10 anti-idiotype fragments were found to have an artificially reduced affinity for NC41 compared to the results obtained using site-directed immobilization via C-terminal thiol residue and from solution equilibrium measurements.

Linear gene fusions of antibody fragments with streptavidin can be linked to biotin labelled secondary molecules to form bispecific reagents.

Monomeric single chain antibody (scFv) fragments lack both the avidity of the bivalent IgG, or (Fab')2 fragment, and the effector functions conferred by the Fc domain. For certain diagnostic or therapeutic applications it may be desirable to link these molecules to other proteins, antibodies, enzymes or peptide ligands, and chemical or recombinant methods have been developed to produce many of these crosslinked reagents. One approach has been to link an antibody fragment to streptavidin which can bind a second biotinylated molecule to create a higher affinity, bifunctional or bispecific molecule.

Influence of mass transfer and surface ligand heterogeneity on quantitative BIAcore binding data. Analysis of the interaction of NC10 Fab with an anti-idiotype Fab'.

The interaction of monovalent Fab fragments of NC10, an antiviral neuraminidase antibody, and the anti-idiotype antibody 3-2G12 has been used as a model system to demonstrate experimentally the influence of non-ideal binding effects on BIAcore binding data. Because the association rate constant for these two molecules was found to be relatively high (about 5 x 10(5) M-1 S-1), mass transfer was recognised as a potential source of error in the analysis of the interaction kinetics. By manipulation of the flow rate and the surface density of the immobilised ligand, however, the magnitude to this error was minimised.

Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero-residue linker a trimer.

Single-chain variable fragments (scFvs) of anti-neuraminidase antibody NC10 were constructed by joining the VH and VL domains with 10-residue (Gly4Ser)2 and five-residue (Gly4Ser) linkers; a zero-residue linker scFv was constructed by joining the C-terminal residue of the VH domain to the N-terminus of the VL domain. The scFv with the 10- and five-residue linkers exclusively formed dimeric antibody fragments (M(r) 52000). These were shown to be bivalent and were able to cross-link two neuraminidase tetramers to form a 'sandwich' type complex; each antigen combining site could also bind an anti-idiotype Fab'.

Identification and minimization of nonideal binding effects in BIAcore analysis: ferritin/anti-ferritin Fab' interaction as a model system.

The interaction of human spleen ferritin with a monoclonal antibody Fab' fragment has been studied as a model system for BIAcore analysis. In particular, the influence of nonideal binding effects has been examined both experimentally and by the theoretical simulation of sensorgram curves. Mass transfer effects were found to have a small but significant influence on the observed binding kinetics of the ferritin/antiferritin Fab' interaction; however, this nonideal behavior could be overcome by systematic manipulation of experimental conditions such as the flow rate and the surface density of the immobilized antigen.

Truth telling and fatal illness.

We argue that if a terminally ill patient wants to know the truth about his or her condition, and the patient's doctor possesses that information, then the doctor is morally obliged to tell the truth. The argument is not based on any supposed right to information (even information about oneself); nor on any supposed moral obligation to tell the truth; nor on any supposed property rights the patient might have. Rather, the argument is based on the very principle usually invoked to license deception: the harm principle (that doctors ought to take that course of action which does least harm to the patient).