Experimentally determined translocation of 134Cs in potatoes. Comparison with the radioecological model ECOSYS-87 and literature data.

The results of a greenhouse experiment on the translocation rate of 134Cs from potato leaves to tubers were compared with calculations of the radioecological model ECOSYS-87 and other literature values. The 134Cs activities applied at three development stages (three pinnate leaves fully developed, onset of flowering, onset of yellowing) to leaves of the plant were taken as starting points for the model to calculate the activity in the tubers at harvest. The default yield in the model was replaced by the experimentally obtained values. The translocation rate measured in the greenhouse experiment was 4 to 14 times higher than the calculations of the model. Some possible reasons for such a high translocation rate, compared with the literature data, are discussed. Based on these comparisons, it is concluded that maximal translocation occurs at the growth stage of flowering of a crop and that the development stage of a crop might be a stronger parameter to describe the time dependency of translocation than the usually applied parameter 'days before harvest'.

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.

Superparamagnetically labelled neutrophils as potential abscess-specific contrast agent for MRI.

In order to evaluate the potential of superparamagnetically labelled neutrophils as inflammation-specific contrast agents for magnetic resonance imaging (MRI), various types of magnetite particles were associated with isolated human neutrophils in vitro and subsequent effects on characteristic cellular functions investigated. Exposure to uncoated magnetite caused strong irreversible cell aggregation, whereas uptake of polystyrene-embedded magnetite microcrystals (Estapor M1-0.70/60) occurred with only minor changes in neutrophil adhesion properties, cellular metabolism, and chemotactic behaviour in vitro. As revealed by MR phantom imaging, Estapor-labelled neutrophils generated visible contrast above a threshold concentration of 1-2 micrograms Fe/g in a tissue-equivalent environment. Intravenous administration of labelled neutrophils to rabbits resulted in local magnetite accumulation up to 2.4 micrograms Fe/g tissue in artificial inflammation sites. Subsequent T2-weighted imaging of an intramuscular abscess clearly demonstrated the expected hypointense area surrounding the typically bright core of inflammation.

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.

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.

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.

Immunoscintigraphic localization of inflammatory lesions: pharmacokinetics and estimated absorbed radiation dose in man.

Five patients with inflammatory lesions received anti-granulocytes murine monoclonal antibody (Mabgc) infused over 5 to 15 min at doses between 3.4 and 5.4 mCi 123I (120 micrograms antibody). Clearance of 123I from blood pool closely fits a biexponential mathematical model with the two effective half-lives 0.73 h and 9.3 h. The spontaneous release of 123I was found to be relatively low in the blood pool. The cumulative urinary excretion of the 123I label over 120 h was in the range of 63% of the totally administered dose and is assumed to represent only a low molecular compound or 123I alone as iodide. Analysis of the label in spleen, liver and red marrow showed that the concentration of label in these tissues remains more or less constant over a period of 20 h after infusion. With data of liver, spleen, red marrow and whole body activity over a period of 24 h, an estimated radiation dose was calculated. Compared with 111In labelled leucocytes, especially in spleen, the absorbed dose is lower by a factor of ten per examination.

Immunoscintigraphic localization of inflammatory lesions: concept, radiolabelling and in vitro testing of a granulocyte specific antibody.

Current nuclear medicine techniques for the localization of inflammatory processes are based on injection of 111In labelled autologous granulocytes which need to be isolated and radiolabelled in vitro before reinjection. A new technique is presented here that obviates the need for cell isolation by the direct intravenous injection of a granulocyte specific 123I labelled monoclonal antibody. In this publication the basic parameters of the antibody granulocyte interaction are described. Antibody binding does not inhibit vital functions of the granulocytes, such as chemotaxis and superoxide generation. Scatchard analysis of binding data reveals an apparent affinity of the antibody for granulocytes of 6.8 X 10(9) l/mol and approximately 7.1 X 10(4) binding sites per cell. Due to the high specificity of the antibody, the only expected interference is from CEA producing tumors.

Immunoscintigraphic localization of inflammatory lesions: clinical experience.

This clinical study was based on the experimental results reported in the two preceding papers, showing that the highly selective affinity of the 123I-anti-CEA monoclonal antibody 47 (123I-Mabgc) for human granulocytes makes this compound suitable for the immunoscintigraphic detection of inflammatory lesions. Forty five patients with suspected infections have been studied after infusion of 4 mCi (148 MBq) 123I-Mabgc corresponding to 120 micrograms labeled protein. No adverse reactions have been seen. Because of the high number of labeled cells, the quality of the images was excellent. SPECT was performed in 15 cases in order to define the extent of the lesion. Infectious foci were usually seen 3-5 h postinjection, but the unimpaired function of the granulocytes guarantees diagnostically relevant examinations over a much longer period of time. Scans were read as being negative if no pathological accumulation of activity was detected after 24 h. The new scanning method is technically easy to perform and provides distinct advantages over other techniques necessitating in vitro labeling of the white blood cells. Therefore, recommended indications are acute infections of unknown origin or extent, especially recurrent episodes of osteomyelitis and infections of joint prostheses.

[125I]amatoxin and anti-amatoxin for radioimmunoassay prepared by a novel approach: chemical and structural considerations.

A novel approach for the production of [125I]amatoxin and anti-amanitin is described. The antigen and the starting material for Bolton Hunter iodination is prepared by periodic acid oxidation of the gamma-delta-dihydroxy-isoleucine side chain of alpha-amanitin followed by reductive amination. The antigen seems to be of low apparent toxicity. Chemical, spectroscopic and immunological evidence suggests that the hapten has a secondary structure similar to alpha-amanitin, but a modified tryptophanyl ring system. The establishment of a clinically useful iodinated radioimmunoassay system is possible, because the ring system seems to be of limited immunological importance.

Radiolabelling of antibodies: methods and limitations.

The development of a labelled antibody for radioimmunoscintigraphic tumor localisation necessitates a major optimisation procedure to carefully balance factors such as radionuclide, anchoring site on the antibody, method of labelling and addition of radioprotective agents. Even under ideal conditions radiolytic and enzymatic degradation of the biologically active antibody cannot be avoided. The resulting radioactive fragments will exhibit organ distribution patterns different from those of the parent antibody and may pose a major obstacle to scintigraphy of certain organs such as liver, kidney and spleen.

Imaging of inflammatory and infectious lesions after injection of radioiodinated monoclonal anti-granulocytes antibodies.

Successful detection of inflammatory lesions by planar scintigraphy and SPECT after injection of iodine-123 labelled monoclonal antibodies directed against human granulocytes (123I-Mabgc) is demonstrated. This new tracer has been compared with indium-111 labelled white blood cells (111In-WBC) in selected patients with proven infectious lesions. Scans were equally positive in all cases, but the methodical advantages of the new marker were obvious, namely, there is no need for cell separation and the images of inflammatory lesions were better defined. In addition, SPECT could be performed with 123I-Mabgc and allowed a better anatomic localization and a three-dimensional description of the lesions. No adverse reactions have been seen. It is concluded, therefore, that 123I-Mabgc is a promising agent for the detection of acute focal inflammatory lesions which may, with advantages, replace 111In-WBC.

Radioimmunoassay for amatoxins by use of a rapid, 125I-tracer-based system.

In this new radioimmunoassay system for determination of amatoxins in urine and plasma, a novel chemical approach is used for antigen and 125I-tracer production, based on a detoxified alpha-amanitin derivative (aldoamanitin). Total assay time, including data processing, is less than 100 min. The lowest detectable concentration is 1 microgram/L for urine, 0.1 microgram/L for plasma. In the clinically significant range, within-run CVs are less than 8%. This new 125I-based assay is a significant improvement over existing 3H technology in terms of speed, precision, and freedom from interference.

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.

[Virologic diagnosis of mumps infection: a solid phase radioimmunoassay for the detection of mumps specific IgM antibodies (MACRIA)]. / Virologische Diagnostik der Mumps-Infektion: ein Festphasen-Radioimmuntest zum Nachweis mumpsspezifischer IgM-antikörper (MACRIA).

A solid phase radioimmunoassay for the detection of mumpsspecific IgM antibodies (MACRIA) is described. In 22 cases of clinically suspected mumps infections confirmed by conventional serological methods, IgM antibodies to mumps virus were detectable. On the other hand, no mumpsspecific IgM antibodies were present in 9 cases of suspected mumps that could not be confirmed by classical serology (seroconversion or significant titer rise in CFT or NT). Neither could IgM antibodies to mumps virus be detected in the sera of 19 rheumatoid factor positive patients nor in those of 20 healthy laboratory personnel. 20 patients from a rubella outbreak who had been found to be positive for antirubella IgM in a comparable MACRIA system showed no IgM response in mumps-MACRIA. In one case of mumps infection during pregnancy where mumps virus was isolated from the salivary glands by throat swab, IgM antibody response was delayed and markedly weaker than in all other patients with a positive IgM response. For this patient, who showed a significant titer rise in NT and seroconversion in CFT to mumps virus, the possibility of reinfection is discussed.

Evidence for the synthesis of a somatomedin similar to insulin-like growth factor I by chick embryo liver cells.

Chick embryo liver cells were incubated in serum-free and hormone-free medium and shown to produce a mitogen starting after the first day in culture. This mitogen was purified by gel filtration and ion-exchange chromatography and identified by several assay systems as a somatomedin similar to human insulin-like growth factor I (IGF-I). This somatomedin stimulates thymidine incorporation into DNA of chick fibroblasts, it reacts in an IGF-specific protein binding assay and it cross-reacts immunologically with antibodies prepared against human IGF-I, but not with those against IGF-II. Its behaviour on ion-exchange chromatography and on sodium dodecylsulfate/polyacrylamide gel electrophoresis is indistinguishable from that of authentic human IGF-I. Concomitantly with the mitogenic activity, chick liver cells produce IGF-binding protein. Addition of purified human IGF-I to the liver cells inhibits production of the binding protein. Chick liver cells may thus be used to study synthesis and regulation of IGF and its binding protein.

The effects of drug which destroy the sympathetic nervous system on the retrograde transport of nerve growth factor.

It has been proposed that the drugs (6-hydroxydopamine, guanethidine, vinblastine) which are known to destroy sympathetic neurons in neonatal animals do so by preventing the accumulation of retrogradely transported nerve growth factor (NGF). It was found, consistent with the proposal, that administration of 6-hydroxydopamine (100 mg/kg s.c.) or vinblastine (0.4 mg/kg s.c.) 16 h prior to the administration of [125I]NGF complete prevented the accumulation of retrogradely transported [125I]NGF in superior cervical ganglia of neonatal rats. Administration of 6-hydroxydopamine or vinblastine to adult rats (where it does not cause sympathetic neuron cell death) did not completely prevent the retrograde transport of NGF, although 6-hydroxydopamine produced an alteration of the time course of accumulation (early times unaffected, later times depressed). The administration of guanethidine to adult rats (50 mg/kg/day) produced a modest decrease in the accumulation of NGF (40-60%). It would appear, however, that this decrease cannot account for the cytotoxic effects of guanethidine since: (1) sub-cytotoxic doses of guanethidine and non-cytotoxic guanidinium blocking agents also produce modest decreases in the retrograde transport in NGF; and (2) the retrograde transport of [125I]NGF is not affected in neonatal animals until after the neurons are clearly damaged. Hence, the data are entirely consistent with the hypothesis that NGF deprivation caused by 6-hydroxydopamine and vinblastine is the mechanism of the cytotoxic effects of these drugs on sympathetic neurons in neonatal animals. Guanethidine destroys sympathetic neurons by some other mechanism.