The use of n-(9-anthroyloxy) fatty acids to determine fluidity and polarity gradients in phospholipid bilayers.

A set of n-(9-anthroyloxy) fatty acid probes (n = 2, 6, 9, 12) have been used to examine gradients in fluorescence polarization, lifetime (tau F), relative quantum yield (phi rel) and positions of emission maxima (lambda max) through bilayers composed of synthetic phospholipids. The fluorophores of these probes report the environment at a graded series of depths from the surface to the centre of the bilayer structure. 1. Polarizations decrease as the fluorophore is moved deeper into the bilayer indicating greater rotational motion of the fluorophore in the hydrocarbon core of the bilayer. 2. The different responses of the probe diphenylhexatriene and the anthroyloxy fatty acids to the action of cholesterol on lipid bilayers are discussed in terms of the orientation of these probes in the bilayer and the types of anisotropic rotational motions which result in depolarization of fluorescence. 3. Stearic acid derivatives which have the fluorophore in the 6-, 9- and 12-positions along the acyl chain have a similar response to solvent polarity as measured by values of lambda max and phi rel in a variety of organic solvents. 4. The position of the emission maximum has little dependence on solvent viscosity, but viscosity does change the degree of vibrational structure seen in the emission spectrum. The vibrational structure itself may be used as an indication of the 'mciroviscosity' gradient in the transverse plane of the bilayer. 5. Values of lambda max, tau F and phi rel indicate that a gradient of polarity exists from the surface to the centre of the bilayer. For dipalmitoyl phosphatidylcholine in the crystalline phase, cholesterol acts to make this polarity gradient shallower.

Green fluorescent antibodies: novel in vitro tools.

We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.

Rapid and precise epitope mapping of monoclonal antibodies against Plasmodium falciparum AMA1 by combined phage display of fragments and random peptides.

We describe an approach for the rapid mapping of epitopes within a malaria antigen using a combination of phage display techniques. Phage display of antigen fragments identifies the location of the epitopes, then random peptide libraries displayed on phage are employed to identify accurately amino acids involved in the epitope. Finally, phage display of mutant fragments confirms the role of each residue in the epitope. This approach was applied to the apical membrane antigen-1 (AMA1), which is a leading candidate for inclusion in a vaccine directed against the asexual blood stages of Plasmodium falciparum. As part of the effort both to understand the function of AMA1 in the parasite life cycle and to define the specificity of protective immune responses, a panel of monoclonal antibodies (MAbs) was generated to obtain binding reagents to the various domains within the molecule. There is a pressing need to determine rapidly the regions recognized by these antibodies and the structural requirements required within AMA1 for high affinity binding of the MAbs. Using phage displaying random AMA1 fragments, it was shown that MAb5G8 recognizes a short linear epitope within the pro-domain of AMA1 whereas the epitope recognized by MAb 1F9 is reduction sensitive and resides within a disulphide-bonded 57 amino acid sub-domain of domain-1. Phage displaying random peptide libraries and mutant AMA1 fragments were employed for fine mapping of the MAb5G8 core epitope to a three-residue sequence in the AMA1 prodomain.