[en] A simple, rapid, and reproducible micromethod for quantification of sulfhydryl (SH) groups generated after reduction of monoclonal antibody (MAb) disulfide bonds with 2-mercaptoethanol (2-ME) is described. The number of SH groups per molecule of antibody in the 2-ME and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. Results were plotted as absorbance at 405 nm vs. cysteine concentration (μg/mL). After subtraction of the background due to Ellman's reagent, a straight-line relationship passing through the origin was obtained. Absorption spectrum of the yellow products was controlled, and no significative differences were found between optical density at 412 nm and 405 nm. Using a small quantity of antibody in the order of 37 μg, the lowest detection limit for cysteine quantification was 0.03 μg. An excellent linear correlation was found between both cysteine concentration and absorbance (r = 0.999), and the mean value of the relative error in the quantification of cysteine from samples was 2.8%. A statistical Student t-test showed an excellent linearity and parallelism between cysteine standard and samples

[en] Reduction of disulfide bonds to sulfhydryl (SH) groups for direct radiolabeling of antibodies for immunoscintigraphic studies of colorectal and other cancers continues to be of considerable research interest. We have developed a general strategy and a versatile computer program for the quantification of the number of SH per molecule of antibody (Ab) generated after the treatment of monoclonal antibodies (MAbs) with reducing agents such as 2-mercaptoethanol (2-ME), stannous chloride (SnCl₂), dithiothreitol (DTT), dithioerythritol (DTE), ascorbic acid (AA), and the like. The program we describe here performs an unweighted least-squares regression analysis of the cysteine standard curve and interpolates the cysteine concentration of the samples. The number of SH groups per molecule of antibody in the 2-mercaptoethanol and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. The linear least-squares method fit the standard data with a high degree of accuracy and with the correlation coefficient r of 0.999. A program has been written for the IBM PC compatible computer utilizing a friendly menu to interact with the users. The package allows the user to change parameters of the assay, to calculate regression coefficients slope, intercept and its standard errors, to perform statistical analysis, together with detailed analysis of variance, and to produce an output of the results in a printed format

[en] Addition of sulfhydryl groups with 2-iminothiolane (2-IT) is an important new method for labelling monoclonal antibodies (mAb) and fragments with ^99mTc. F(ab')₂ fragments were prepared by digestion of 1B7.11 and BCD-F9 with pepsin. Optimal conditions for labelling 20-100 μg mAb or F(ab')₂ involved a 2000:1 molar ratio of 2-IT: protein in phosphate buffer pH 7.4 for 30 min followed by addition of^99m Tc-pertechnetate and stannous glucoheptonate. Recovered yields were >70% and radiochemical purities were >90% with a total preparation time of <90 min

[en] The mechanism of photochemical reactions of radicals RS₂, RS/M/H and RS/M/SR formed in the low-temperature photolysis of hydrocarbon solutions of mercaptans has been investigated. The quantum yield of such reactions was also determined. (author)

[en] Chinese hamster V79 fibroblasts were irradiated in the gas explosion apparatus and the chemical repair rates of the oxygen-dependent free radical precursors of DNA double-strand breaks (dsb) and lethal lesions measured using filter elution (pH 9.6) and a clonogenic assay. Depletion of cellular GSH levels, from 4.16 fmol/cell to 0.05 fmol/cell, by treatment with buthionine sulphoximine (50 μmol dm^-3; 18 h), led to sensitization as regards DNA dsb induction and cell killing. This was evident at all time settings but was particularly pronounced when the oxygen shot was given 1 ms after the irradiation pulse. A detailed analysis of the chemical repair kinetics showed that depletion of GSH led to a reduction in the first-order rate constant for dsb precursors from 385 s^-1 to 144 s^-1, and for lethal lesion precursors from 533 s^-1 to 165 s^-1. (Author)

No abstract available

[en] A method for the direct ^99mTc-labeling of antibodies by dithionite reduction was developed. Among three murine monoclonal IgG1 and one human polyclonal IgG (hIgG) antibodies tested, hIgG was the most quickly reduced by dithionite. These differences may reflect the reactivities of antibody disulfide bonds toward the oxidation products of dithionite. By optimizing reduction conditions to generate enough free sulfhydryl groups, it was possible to radiolabel human IgG and monoclonal antibody 170 with ^99mTc with a 90% monomeric antibody efficiency. The process avoided colloid formation. In contrast, about 0.1 sulfhydryl groups per antibody molecule, less than 1% of the possible 36, were detected after treatment with ascorbate (up to 35,000:1 molar ratio) at room temperature for 1 h for the antibodies tested. Sulfhydryl groups generated in antibodies were estimated using a new method: 5-iodoacetamidofluorescein-labeled antibodies quantitated by size exclusion HPLC. Ascorbate was found to prevent antibody aggregate formation in cysteine-challenged samples

[en] Purpose: Aberrant architecture of the tumor vasculature and temporal fluctuations in blood flow can result in tumor hypoxia. The aim of this study was to classify tumor hypoxia based on distance to blood vessels, and to characterize its biologic significance by determining levels of nonprotein sulfhydryls (NPSH) in hypoxic regions located proximally and distally to tumor blood vessels. Methods and Materials: A dual fluorescence method was developed for the spatial colocalization of the vasculature and hypoxia in frozen sections from SiHa cervical carcinoma xenografts. A parallel section was stained with the sulfhydryl stain mercury orange. Composite fluorescence images were generated by imaging and tiling individual fields of view into 2D image arrays. Image arithmetic techniques were combined with feature-based image segmentation to characterize expression of NPSH as a function of the hypoxic tumor microenvironment. Results: NPSH levels were higher in hypoxic areas of the SiHa xenografts (15.1±0.5 vs. 13.5±0.5 integrated optical density [IOD], p<0.03). When tumor hypoxia was classified by distance to the nearest visible blood vessel, significantly higher NPSH levels were found in hypoxic regions close to blood vessels than in regions at a distance from blood vessels. Conclusion: The results of this study indicate differential expression of NPSH levels in regions of hypoxia that are proximal or distal to blood vessels in SiHa tumors

No abstract available

[en] Using the lipophilic chelating agent, acetylacetone, red cells have been radiolabelled with the short-lived, generator-produced isotope, sup(113m)In. Following re-injection of these labelled cells, red cell volume has been measured and compared with corresponding values using sup(99m)Tc labelled red cells in 18 patients, and with ⁵¹Cr labelled red cells in five patients. Sup(99m)Tc slightly overestimated red cell volume in relation to sup(113m)In, but ⁵¹Cr values were identical to sup(113m)In values. There was a close correlation between splenic red cell pool measured with sup(99m)Tc and with sup(113m)In. It was concluded that the intracellular stability and gamma emission of sup(113m)In make this isotope a superior alternative to sup(99m)Tc and ⁵¹Cr in measurements of red cell volume and splenic red cell pool. (author)