Placental transmission of thyroid-stimulating immunoglobulins.

Long-acting thyroid stimulator (LATS) and LATS protector (LATS-P) were assayed at or near delivery in serum from 18 pregnant women with a history of past or present thyrotoxicosis. The results suggested that neonatal thyrotoxicosis may be predicted prenatally if maternal serum LATS and LATS-P concentrations near delivery are above certain levels.     

Evaluation of immunoglobulins from plant cells.

Expression of cDNA constructs encoding full-length mouse immunoglobulin chains with their native leader sequences or fusion constructs substituting the native leader with a pre-pro sequence derived from Saccharomyces cerevisiae yielded blocked N-termini on the gamma chain or the correct amino terminal sequence on the mature kappa chain. Lectin binding assays revealed that assembled immunoglobulin complexes contained a glycosylated heavy chain. The attached glycan was resistant to digestion by endoglycosidase H and its lectin binding pattern was distinguishable from that of the mammalian glycan. The results indicated processing of the immunoglobulin carbohydrate in the tobacco Golgi to yield a complex oligosaccharide. Secretion of antibody by protoplasts isolated from regenerated transgenic plants or from suspension callus cells was demonstrated by pulse-chase labeling experiments. When purified, the tobacco-produced antibody was found to possess the antigen binding and catalytic properties of the murine monoclonal antibody. Kinetic parameters (Km, Ki, Vmax, and kcat) of the tobacco-derived antibody were comparable to those of the mouse-derived antibody. The results in general show that the endomembrane system of tobacco cells possesses cognate mechanisms for the recognition of diverse leader sequences. These signals can be used to initiate the assembly, processing, and secretion by plant cells of complex foreign proteins.     

Immobilization of immunoglobulins on silica surfaces. Stability.

The development of new immunosensors based on surface-concentration-measuring devices requires a stable and reproducible immobilization of antibodies on well-characterized solid surfaces. We here report on the immobilization of immunoglobulin G (IgG) on chemically modified silica surfaces. Such surfaces may be used in various surface-oriented analytical methods. Reactive groups were introduced to the silica surfaces by chemical-vapour deposition of silane. The surfaces were characterized by ellipsometry, contact-angle measurements and scanning electron microscopy. IgG covalently bound by the use of thiol-disulphide exchange reactions, thereby controlling the maximum number of covalent bonds to the surface, was compared with IgG adsorbed on various silica surfaces. This comparison showed that the covalently bound IgG has a superior stability when the pH was lowered or incubation with detergents, urea or ethylene glycol was carried out. The result was evaluated by ellipsometry, an optical technique that renders possible the quantification of amounts of immobilized IgG. The results outline the possibilities of obtaining a controlled covalent binding of biomolecules to solid surfaces with an optimal stability and biological activity of the immobilized molecules.     

Phylogenesis of the general structure of immunoglobulins.

The study of the general structure of macroglobulins and 7S (IgG) immunoglobulins of frog and tortoise by relaxation methods shows the rotational correlation time of 7S immunoglobulins to be 67–68 nsec whereas that of macroglobulins of frog is 135 nsec and of tortoise is 103 nsec. Experimental values of rotational correlation time for 7S immunoglobulins are close to those calculated for the model of this molecule approximated by the rigid rotational ellipsoid and lower than those estimated for macroglobulins. This indicates that frog and tortoise 7S immunoglobulins have a fairly compact general structure with no marked intramolecular rotational freedom for high-molecular fragments, whereas macroglobulins possess it to a limited extent. It is seen from our evidence on the rigidity of 7S immunoglobulins and the limited flexibility of macroglobulins in amphibia and reptiles, as compared to previous data on the limited flexibility of carp macroglobulins and on the pronounced flexibility of IgG and IgM of mammals, that the general structure becomes more flexible on passing from lower vertebrates to representatives of a more recent class of mammals. The reported increased flexibility of immunoglobulins accompanied by the progressive evolution of species is likely to provide one of the first indications of the possible directed selection in the course of molecular evolution.     

Antibody-antigen complex formation with immobilized immunoglobulins.

We have investigated the complex formation between an immobilized monoclonal antibody and antigens that differ in size about 50-fold. As a model system, we used an iodinated progesterone derivative and a progesterone-horseradish peroxidase conjugate as tracer and a monoclonal antibody as binding protein. The antibody was immobilized by four different methods: physical adsorption, chemical binding, and binding via protein G in the absence or presence of a protective protein (gelatin). These investigations have shown that the performance of competitive immunoassays is determined by a combination of factors: (a) the relative size of the analyte and the tracer, (b) the antibody density on the solid matrix, (c) the method of immobilization of the antibody, and (d) the binding constants between antibody-analyte and antibody-tracer. All of these interactions have to be considered in designing an optimal immunoassay. The smaller antigen can form a 3- to 35-fold higher maximal complex density than the larger antigen. Dose-response curves are less affected by the size of the tracer than by the binding constant with the antibody. A large enzyme tracer with a relatively low binding constant can, therefore, provide a more sensitive assay. On the other hand, the increase in complex density achieved with a smaller tracer yields a higher signal that in turn can provide a better signal-to-noise ratio in highly sensitive competitive solid-phase immunoassays. We have suggested a model for antibody immobilization that accounts for the interdependence of tracer size, complex formation, and antibody density. The methods described can be used to design and optimize immunoassays of predefined performance characteristics. The results are particularly useful for converting radioimmunoassays to enzyme immunoassays.