No abstract

Background: Type 1 diabetes mellitus results from destruction of the pancreatic insulin-producing beta cells by a chronic autoimmune process. Methods are needed for the detection of circulating autoantibodies to glutamic acid decarboxylase (GAD65), a major marker of this process. Methods: Streptavidin-coated microtiter plates were incubated with biotinylated GAD65, and after incubation with serum samples from patients with type 1 diabetes mellitus and control individuals, europium-labeled GAD65 was added. After washing steps, the delayed fluorescence was measured in duplicate in a fluorometer. Samples collected from 100 patients with newly diagnosed type 1 diabetes mellitus and 100 healthy controls were measured by the new assay and by a radiobinding assay. Results: The detection limit of the new assay was 1.49 WHO units/mL, the calibration curve was linear to 4 140 WHO units/mL, and no hook effect was observed up to 41 400 WHO units/mL. The intraassay CV was 2.1-6.3% over the calibration range. For patient serum samples, the intraassay, interassay, and total CVs were 5.4 -7.0%, 9.8 -13%, and 12-14%, respectively. Compared with conventional radioimmunologic methods, the analytical range was broader and the analysis time required to

The anti-17␤-estradiol antibody 57-2 has been a subject for several protein engineering studies that have produced a number of mutants with improved binding properties. Here, we generated a set of 16 antibody 57-2 variants by systematically combining mutations previously identified from phage display-derived improved antibody mutants. These mutations included three point mutations in the variable domain of the light-chain and a heavy-chain variant containing a four-residue random insertion in complementarity determining region CDR-H2. The antibody variants were expressed as Fab fragments, and they were characterized for affinity toward estradiol, for cross-reactivity toward three related steroids, and for dissociation rate of the Fab/estradiol complex by using time-resolved fluorescence based immunoassays. The doublemutant cycle method was used to address the cooperativity effects between the mutations. The experimental data were correlated with structural information by using molecular modeling and visual analysis of the previously solved antibody 57-2 crystal structures. These analyses provided information about the steroidbinding mode of the antibody, the potential mechanisms of individual mutations, and their mutual interactions. Furthermore, several combinatorial mutants with improved affinity and specificity were obtained. The capacity of one of these mutants to detect estradiol concentrations at a clinically relevant range was proved by establishing a time-resolved fluorescence based immunoassay.Keywords: Antibody; estradiol; mutation; site-directed mutagenesis; steroidThe capability of antibodies to specifically recognize an immense variety of other molecules is widely used in life sciences. For example, immunoassays that are commonly used for the quantitation of the concentrations of specific components in complex molecular mixtures such as blood are based on the recognition properties of antibodies. However, the generation of high-quality antibodies for some antigens is challenging. An example of this type of molecule is the steroid hormone 17␤-estradiol (E2), which consists of a hydrophobic steroid skeleton containing only two functional hydroxyl groups (Fig. 1). Concentration of E2 in the blood is low, and a number of steroids with closely related structures are present in the circulation, many of them in much higher concentration than E2. Therefore, the antibody used as a binder in an E2 immunoassay needs to have high affinity and good specificity. Currently, sufficiently good monoclonal antibodies are not available, and the E2 immunoassays are based on polyclonal antibodies.We have previously cloned three murine antibodies raised against 17␤-estradiol-6-CMO conjugate (Pajunen et al. 1997). One of these is known as antibody 57-2, and it has a moderate affinity toward E2, an excellent capacity to disReprint requests to: Urpo Lamminmäki, Department of Biotechnology, University of Turku, Tykistökatu 6A, 20520 Turku, Finland; e-mail: urpo.lamminmaki@utu.fi; fax: 358-2-3338050.Abbreviations: E2, ...