Towards amperometric immunosensor devices.

In contrast to optical immunosensors, the electrochemical detection of an immunanalytical reaction does require a labeling, but allows an easier discrimination of specific and non-specific binding. We present a concept and first results for a multivalent amperometric immunosensor system which is based on silicon technology. The capture molecule streptavidin, covalently immobilized on silica, allows the immobilization of biotinylated antigens at a defined density. A nanostructured gold electrode serving as a stable network of nanowires is expected to be beneficial for the electrochemical detection of bound ferrocene-labeled antibody molecules. The results presented focus on site-specific immobilization of streptavidin on silica and reduction of non-specific binding of proteins.

In vivo evaluation of magnetite nanoparticles for use as a tumor contrast agent in MRI.

Magnetite nanoparticles, coated by three different artificial polypeptides, were conjugated to an antibody specific to the carcinoembryonic antigen (CEA). To protect the particles from fast blood elimination, the coats were modified by various sugars, polyethyleneglycol, albumin, and sialoproteins, respectively. The protective effect was determined by using a specific in vitro test and by analyzing the biodistribution of the nanoparticles in nude mice grafted with CEA-tumors. In particular, a prolongation of the blood circulation time has been expected, if a natural modifier is attached to the coated nanoparticles. Although the elimination rate could hardly be decreased by any modifiers, the tumor accumulation is slightly improved by using the specific sialoprotein glycophorin B. The usefulness of nanoparticles as image contrast agents is probably limited by their microdistribution within the tumor tissue. The requirements for a contrast agent to be highly tissue specific are discussed.

Use of a biotinyl-estradiol derivative to demonstrate estradiol-membrane binding sites on adherent human breast cancer MCF-7 cells.

A biotinyl-derivative of 17 beta-estradiol has been used to demonstrate a site of recognition and binding of estradiol located on the plasma membrane of human breast cancer MCF-7 cells by using the biotin/avidin-FITC system. The specificity of this binding has been shown by a displacement of the fluorescent label by 17 beta-estradiol. No displacement was observed when testosterone was added. Quantification of this phenomenon has been shown by laser scanning cytometry while preserving the cells adhesiveness to their growth support as well as their membrane integrity. An analysis by confocal laser scanning microscopy suggested that the fluorescence distribution on MCF-7 cells treated with estradiol-biotin was on the cell periphery. The results obtained are in favour of the recognition and binding site of 17 beta-estradiol located on the plasma membrane of MCF-7 cells and they would indicate that the biological activity of estradiol, among others, could be initiated by an interaction with the membrane.

Antibody-magnetite nanoparticles: in vitro characterization of a potential tumor-specific contrast agent for magnetic resonance imaging.

Target-specific superparamagnetic contrast agents may allow the localization of specific tissues such as tumors by magnetic resonance imaging (MRI). In this report the preparation and in vitro characterization of tumor-specific superparamagnetic particles (SMP) are described. Particles of uniform size (9.6 +/- 0.8 nm) were prepared from an alkaline solution of ferric and ferrous ions and isolated by differential centrifugation. The resulting nanoparticle suspension is stabilized in buffer using a polypeptide coat to which a monoclonal antibody, specific to carcinoembryonic antigen (CEA), was covalently attached at the hinge region. The resulting anti-CEA SMP have a hydrodynamic radius of less than 50 nm, and specifically bind to CEA in vitro. The visualization of epitopes, present on a cell surface in very low density as expected for tumor antigens or receptors, may be achieved due to the high R2 relaxivity of 300 L mmol-1s-1 of the contrast agent described here. Furthermore, the polypeptide coat chosen provides an ideal platform for the attachment of biological modifiers needed for the reduction of the antigenicity and blood clearance rate of anti-CEA SMP.

Covalent immobilization of avidin on glassy carbon electrodes as the basis for multivalent biosensors.

One of the most crucial steps for the successful construction of a biosensor is the appropriate and reproducible coupling of the biological part (e.g. enzyme, antibody) to the inorganic moiety of the device (e.g. electrode, microchip). In this paper three methods of immobilization of avidin to a glassy carbon electrode are described. Depending on the type of immobilization, avidin may lose its biological activity as determined by an enzyme immunoassay, using biotinylated reagents. If avidin is covalently bound to the glassy carbon electrode via the bridge molecule 4.4'-diaminodiphenylamine, the biological activity is retained. About 1.5 pmol of avidin can be bound to the electrode (3 mm in diameter), resulting in a nearly complete monolayer of protein.

A lipophilic complex with 186Re/188Re incorporated in liposomes suitable for radiotherapy.

Liposomes with a 70 nm diameter were made by the detergent removal technique on a gel filtration column. The complex oxodichloroethoxy-bis-(triphenylphosphine)rhenium(V)=(Rephos) was irradiated by neutrons in the reactor at PSI. 45 +/ 5% of the radioactive complex was incorporated into the bilayer of the lipsomes during the liposome formation. The stablility of these radioactive liposomes was tested by dialysis: a loss of 40% of the radioactivity identified as perrhenate was observed after 8 days. Addition of the antioxidant ascorbic acid diminished the loss to 20%. Such liposomes carrying the lipophilic radioactive Re-complex can potentially be used in beta-radiotherapy. The gamma-lines of the two rhenium isotopes are helpful for localizing them in therapy controls by a gamma-camera, a big advantage compared to other nuclides proposed for therapy (e.g. 90Yttrium).

Biotinyl-estradiol derivatives in enzyme immunoassays: structural requirements for optimal antibody binding.

The use of the avidin/biotin complex in immunoassays is well documented. No comprehensive studies, however, are available on the structural requirements of the linkage between biotin and small molecules to get an optimal antigen-antibody interaction. We have synthesized seven different biotinylated estradiol derivatives. They were evaluated in an antibody- and in an antigen-immobilized enzyme immunoassay system. All three derivatives lacking a spacer group were useless for use in immunoassays, demonstrating the importance of a long distance between the biotin- and estradiol-moiety. In addition, the chemical structure of the linkage at the site of attachment to the steroid skeleton is very important for the antibody recognition: it may either be rigid but identical to that one used in the immunogen (6-carboxymethyloxime), or must be structurally flexible as exemplified by a 6-amido-linkage. A rigid structure (hydrazone) different from that of the immunogen absolutely prevents antibody binding.

Treatment of antibodies to reduce non-specific binding in immunoassays using the avidin-biotin complex.

Three different methods to reduce non-specific binding of antibodies to avidin coated plates in an immunoassay are described and compared: The use of a blocking agent, antiserum adsorption on avidin-agarose and antiserum adsorption to avidin by a so called pre-incubation method. The pre-incubation method proved to be highly efficient, fast and simple. Further applications of this method to different immunoassay systems are proposed in the discussion section.

Antigen- versus antibody-immobilized ELISA procedures based on a biotinyl-estradiol conjugate.

A biotinyl-6 alpha-estradiol derivative (Bio-E2) was synthesized and used as the key component in antigen- and antibody-immobilized ELISA techniques, and the relative merits of the two methods were compared. A precise and reproducible antigen-immobilization was achieved in avidin-coated microtiter plates with Bio-E2. This assay, when completed by the incubation with primary antibody and second antibody-peroxidase conjugate, has a very low detection limit (6 pg/ml estradiol) but required a long incubation time with primary antibody to reach equilibrium. At non-equilibrium conditions, using a high antibody concentration, the assay could be very fast and sensitive. In the antibody-immobilized assay, the Bio-E2 was added to compete with the estradiol present in the calibrator or sample and visualized with a streptavidin-peroxidase conjugate. The detection limit is higher (34 pg/ml), but the specificity was superior and the incubation time to reach equilibrium shorter as compared to the antigen-immobilized assay. Therefore, the antibody-immobilized assay appeared to be ideal for the classical ELISA technique, whereas the antigen-immobilized method seemed to be best suited for automated assay systems using antibody in excess.

Prevention of bridge binding in immunoassays: a general estradiol tracer structure.

Iodinated estradiol tracers were synthesized with three different bridges connecting the radiolabelled moiety to the steroid core: Hemisuccinate, carboxymethyloxime and amide. Taking these iodinated tracers in combination with ten antibodies raised against estradiol-6-CMO-albumin, titers and slopes of calibration curves have been compared to the corresponding data using a 3H tracer. The data indicate that the tracer with the amide bridge is recognized similarly to the tritiated estradiol by all antibodies tested, whereas the two other iodinated tracers exhibit substantial bridge binding. The results suggest that the amide tracer structure can generally be used to improve the quality of estradiol antibodies suffering from bridge binding effects.

Prevention of bridge binding effects in haptenic immunoassay systems exemplified by an iodinated radioimmunoassay for melatonin.

Antisera used in immunological assay systems for small molecular weight substances are routinely prepared by coupling the hapten to a carrier protein via a chemical linker. Often this bridge is partly recognized by the antibody, resulting in reduced sensitivity when an identically structured tracer (e.g. iodine-labelled) is used. Historically, the problem was solved by changing the linking structures in the tracer. An alternative way is exemplified by the development of a very sensitive and specific iodinated radioimmunoassay for melatonin. This new approach involves the design of a linkage identical in the tracer and the antigen that is both very short and closely resembling the structure of the analyte itself.

Chronic granulomatous disease: effect of sulfamethoxazole/trimethoprim on neutrophil microbicidal function.

Normal and chronic granulomatous disease (CGD) neutrophils accumulated sulfamethoxazole (SMX) 3-fold and trimethoprim (TMP) 14-fold, possibly through a non-ionic diffusion and pH-partition mechanism. CGD neutrophils incubated with SMX/TMP after phagocytosis of S. aureus killed the bacteria. These findings explain the clinically observed beneficial effect of SMX/TMP in the treatment of infections in CGD and in other conditions characterized by impaired phagocyte microbicidal capacity.