One of the most basic questions in single-molecule microscopy concerns the accuracy with which the location of a single molecule can be determined. Using the Fisher information matrix it is shown that the limit of the localization accuracy for a single molecule is given by, lambda(em)/2pi n(a) square root of gammaAt, where lambda(em), n(a), gamma, A, and t denote the emission wavelength of the single molecule, the numerical aperture of the objective, the efficiency of the optical system, the emission rate of the single molecule and the acquisition time, respectively. Using Monte Carlo simulations it is shown that estimation algorithms can come close to attaining the limit given in the expression. Explicit quantitative results are also provided to show how the limit of the localization accuracy is reduced by factors such as pixelation of the detector and noise sources in the detection system. The results demonstrate what is achievable by single-molecule microscopy and provide guidelines for experimental design.

In antibodies, a heavy and a light chain variable domain, VH and VL, respectively, pack together and the hypervariable loops on each domain contribute to binding antigen. We find, however, that isolated VH domains with good antigen-binding affinities can also be prepared. Using the polymerase chain reaction, diverse libraries of VH genes were cloned from the spleen genomic DNA of mice immunized with either lysozyme or keyhole-limpet haemocyanin. From these libraries, VH domains were expressed and secreted from Escherichia coli. Binding activities were detected against both antigens, and two VH domains were characterized with affinities for lysozyme in the 20 nM range. Isolated variable domains may offer an alternative to monoclonal antibodies and serve as the key to building high-affinity human antibodies. We suggest the name 'single domain antibodies (dAbs)' for these antigen binding demands.

We have engineered the Fc region of a human immunoglobulin G (IgG) to generate a mutated antibody that modulates the concentrations of endogenous IgGs in vivo. This has been achieved by targeting the activity of the Fc receptor, FcRn, which serves through its IgG salvage function to maintain and regulate IgG concentrations in the body. We show that an IgG whose Fc region was engineered to bind with higher affinity and reduced pH dependence to FcRn potently inhibits FcRn-IgG interactions and induces a rapid decrease of IgG levels in mice. Such FcRn blockers (or 'Abdegs,' for antibodies that enhance IgG degradation) may have uses in reducing IgG levels in antibody-mediated diseases and in inducing the rapid clearance of IgG-toxin or IgG-drug complexes.

Preclinical tests of therapeutic antibodies are frequently carried out in mice to evaluate pharmacokinetics and efficacy. However, the observation that mouse IgG are cleared rapidly from the human circulation suggests that mice may not always be an ideal model. The Fc receptor, FcRn, regulates the serum half-lives of IgG in mice and most likely has a similar function in humans. In the current study we have carried out an extensive analysis of the interaction of the human or mouse forms of FcRn with IgG from various species using surface plasmon resonance. We show that in contrast to mouse FcRn, human FcRn is surprisingly stringent in its binding specificity for IgG derived from different species. Human FcRn binds to human, rabbit and guinea pig IgG, but not significantly to rat, bovine, sheep or mouse IgG (with the exception of weak binding to mouse IgG2b). In contrast, mouse FcRn binds to all IgG analyzed. The lack of binding of human FcRn to mouse IgG1 has been confirmed using transfectants that have been engineered to express human FcRn on the cell surface. Our results provide a molecular explanation for the enigmatic observation that mouse IgG behave anomalously in humans. These studies have implications for the successful application of therapeutic antibodies.

The MHC class I-related receptor, FcRn, mediates the transfer of maternal gamma globulin (IgG) to young rodents, primarily via intestinal transcytosis, and this provides humoral immunity for the first few weeks after birth. In a previous study, the site of mouse IgG1 (mIgG1) with which FcRn interacts has been mapped using recombinant wild-type and mutated Fc-hinge fragments. The site encompasses residues at the CH2-CH3 domain interface of Fc (Ile253, His310, Gln311, His433 and Asn434) and the same amino acids are involved in regulating the pharmacokinetics of the Fc-hinge fragments. This suggests that in addition to its known function, FcRn might also play a role in IgG homeostasis. Consistent with this hypothesis, in this study, we demonstrate that FcRn alpha-chain mRNA is present not only in neonatal brush border but also in other tissues of adult animals (liver, lung, spleen and endothelial cells). In addition, analysis of the pharmacokinetics of mouse Ig/Fc-hinge fragments in genetically manipulated mice that are deficient in the expression of FcRn demonstrates that the beta-phase half-lives are abnormally short. These findings suggest that FcRn is involved in IgG homeostasis.