The effect of earthworms on copper fractionation of freshly and long-term polluted soils.

We investigated the effects of earthworm activity on the bioavailability of Cu in soil. The bioavailable fraction was estimated using sequential extraction, and the results of diethylenetriaminepentaacetic acid (DTPA) extraction were analyzed for comparison. Changes in the Cu fraction were compared in Cu-spiked soil (high bioavailability) and long-term polluted field soil (low bioavailability) with approximately equivalent total Cu concentrations. Earthworm activity decreased the bioavailable fraction in the Cu-spiked soil, where earthworm body Cu concentrations did not affect the bioavailable fraction. Soil pH was not a factor in the bioavailability differences between soils with and without earthworms in this study. The bioavailable fraction appears to be more heavily affected by biological and physical mechanisms than by soil pH. The two extraction methods showed different trends; the bioavailable fraction method was better than DTPA extraction, because the former gives clear insight into the aging process of Cu in soil.

Clinical-scale radiolabeling of a humanized anticarcinoembryonic antigen monoclonal antibody, hMN-14, with residualizing 131I for use in radioimmunotherapy.

UNLABELLED: Radiolabeling of monoclonal antibodies (mAbs) with an intracellularly trapped form of (131)I (residualizing (131)I) involves radioiodinating a small molecular entity, conjugating it to the mAb, and purification. Column purifications are impractical during procedures involving multi-gigabecquerel levels of radioactivity. The goal of this study was to develop a simple, remote, "1-pot" method of radiolabeling and purification for the scaled-up radioiodination of a humanized anti-carcinoembryonic antigen (CEA) mAb, humanized MN-14 (hMN-14; labetuzumab), with an optimized residualizing (131)I moiety, (131)I-IMP-R4. IMP-R4 is MCC-Lys(MCC)-Lys(X)-d-Tyr-d-Lys(X)-OH, where MCC is 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl and X is 1-((4-thiocarbonylamino)benzyl)-diethylenetriaminepentaacetic acid. METHODS: An IODO-GEN-based remote labeling system was used. IMP-R4 was radioiodinated (0.13 mumol per 3.7 GBq of (131)I) at a pH of 7.0-7.4 and conjugated to disulfide-reduced hMN-14 after quenching of unused reactive (131)I. The product was purified by stirring for 5 min with a 20% (w/v) suspension of an anion-exchange resin and sterilely filtered into a sealed vial. Human serum albumin was added at a final concentration of 1%-2.5%. Immunoreactivity was determined by mixing with CEA and determining the complexation level by size-exclusion high-pressure liquid chromatography. Two control radiolabelings, either with unreduced hMN-14 or with IMP-R4 omitted, also were performed. RESULTS: In 18 radiolabelings with (131)I in the range of 2.04-4.81 GBq (55-130 mCi), yields of 59.9% +/- 7.9% (mean +/- SD) at specific activities of 200 +/- 26 MBq/mg (5.4 +/- 0.7 mCi/mg) were obtained, with > or =95% of the radioactivity being associated with hMN-14 and with < or =4% aggregation. Similar yields were obtained in a subset of radiolabelings (n = 7) with >3.7 GBq of (131)I. The immunoreactivities of the preparations were typically >95%. Nonspecific incorporation in the absence of IMP-R4 was 0.5%, whereas that obtained with unreduced IgG was approximately 8%, possibly because of conjugation of IMP-R4 at lysine sites. The process also removed >99% of the quenching reagent used. Radiolabelings performed with freshly prepared solutions or lyophilized preparations produced similar yields, a result that suggested the option for a single-use kit design. CONCLUSION: Efficient removal of (131)I-IMP-R4 and quenched (131)I by 5 min of stirring with anion-exchange resin renders a multi-gigabecquerel-level preparation of (131)I-IMP-R4-hMN-14 safe, convenient, and practical.

Automated preparation of 188Re-labeled radiopharmaceuticals for endovascular radiation therapy.

We have developed an automated system for the preparation of highly concentrated 188Re-perrhenate, diethylenetriamine pentaacetic acid (DTPA) and mercaptoacetyltriglycine (MAG3). The three procedural steps include concentration of 188Re-perrhanerate, chelation and purification and sterilization. The steps are operated by a small micro-controller. The eluted 188Re-perrhenate of 15 GBq/18 ml from 37 GBq 188W/188Re-generator was concentrated to 1.2 ml in 10 +/- 0.5 min with a recovery yield of 95 +/- 1.5%. We obtained the highest radiochemical yield of 95.4 +/- 2.8% and 98.5 +/- 1.2% for 188Re-DTPA and MAG3 at the oil bath temperatures of 95-97 degrees C and 93-97 degrees C, respectively.

Preparation of indium-115-labeled diethylenetriaminetetraacetic acid monoacetamide peptides purified by 8-hydroxyquinoline.

In this communication we describe a novel procedure for the preparation and purification of diethylenetriaminepentaacetic (DTPA)-acylated and (115)In(3+)-labeled oligopeptides using 8-hydroxyquinoline for the removal and quantification of nonbound indium ions. First the N(alpha)- or N(alpha),N(epsilon)-DTPA oligopeptides containing C-terminal KDEL signal motif were produced by solid-phase synthesis. For this the free carboxyl group of DTPA dianhydride was activated in situ for a short period of time yielding a major product. Reversed-phase HPLC-purified DTPA oligopeptides were labeled with (115)In(3+) in aqueous buffer solution at pH 3.8. For the removal as well as for the detection of uncoordinated (115)In(3+) ions we have utilized the (115)In(3+) complex-forming ability of 8-hydroxyquinoline in chloroform. Following an optimized extraction procedure the free indium ion content was measured by spectrophotometry in the organic phase. Data obtained by this method and verified by total-reflection X-ray fluorescence spectroscopy and thin-layer chromatography demonstrated that free (115)In(3+) could be efficiently removed and sensitively detected in the presence of DTPA oligopeptide chelator. No release of (115)In(3+) from its DTPA complex was observed. This method could be useful for the preparation of indium complexes of peptides and perhaps proteins containing a DTPA moiety and nonradioactive isotope ligand.

Thiol-reactive, luminescent Europium chelates: luminescence probes for resonance energy transfer distance measurements in biomolecules.

Lanthanide chelates have recently been shown to be extremely promising luminescence probes for distance measurements in biomolecules using luminescence resonance energy transfer measurements [P. R. Selvin, T. M. Rana, and J. E. Hearst (1994) J. Am. Chem. Soc. 116, 6029-6030; P. R. Selvin, and J. E. Hearst (1994) Proc. Natl. Acad. Sci. USA 91, 10024-10028]. In this work we describe simple procedures for preparing highly fluorescent thiol-reactive europium chelates. These new compounds contain a uv-absorbing coumarin group which sensitizes europium emission, diethylenetriaminepentaacetic acid or triethylenetetraaminehexaacetic acid groups which provide europium chelating function, and a pyridyl disulfide group which allows specific modification of thiol groups. These reagents can be used to label proteins at Cys residues or synthetic oligonucleotides which contain thiol groups. Modification can be reversed easily by treatment with a reducing agent (dithiothreitol). Luminescence energy transfer between these new chelates and CY5 fluorochrome attached to the opposite ends of 15-bp double-stranded DNA was measured to test their usefulness for distance measurements in macromolecules. The distance measured between the chelate (donor) and CY5 (acceptor) was in the range expected for the length of 15-bp DNA. The stability of europium chelates and their conjugates with a protein, the precision of distance measurements using these chelates, possible errors due to intramolecular energy transfer, and the modulation of the R0 value with deuterium oxide were tested. The results obtained fully confirmed the great potential of these new probes for sensitive, simple, and precise distance measurements in biomolecules using luminescence resonance energy transfer.

Purification of p-nitrobenzyl C-functionalized diethylenetriamine pentaacetic acids for clinical applications using anion-exchange chromatography.

A general process for the purification of large quantities (5-10 g) of p-nitrobenzyl C-functionalized diethylenetriamine pentaacetic acids is reported. The method of choice to achieve purification for clinical applications is anion-exchange chromatography.