Biliary metabolites of testosterone and testosterone glucosiduronate in the rat

A mixture of testosterone-4-¹⁴C and testosterone-1,2-³H-17-glucosiduronate was intraperitoneally administered into male and female rats with bile fistulas. Biliary metabolites were separated and purififd by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the blue. 5β-Androstane-3α, 17β-diol was found principally in monoglucosiduronate fraction and was produced preferentially from the injected conjugate in both sexes. Very marked sex differences from the injected conjugate in both sexes. Very marked sex differences were observed in the following metabolites: Androsterone was present only in the female as monoglucosidironate, which was preferentially derived from testosterone. 5α-Androstane-3α,17β-diol was identified in both monoglucosiduronate and diconjugate fractions of the female, which was formed significanrly more from the conjugate than testosterone. These findings provide evidence that testosterone glucosiduronate could be converted directly into 5α-steroids as well as 5β-ones $\text{in}$$\text{vivo}$. In marked contrast, the major portion of testosterone was metabolized to polar steroids in the male.     

The use of CdTe quantum dot fluorescent microspheres in fluoro-immunoassays and a microfluidic chip system.

Polystyrene fluorescent microspheres prepared by deposition of CdTe quantum dots (QDs) are used in an immunoassay in this study. CdTe QDs/polyelectrolyte multilayers on the surface of polystyrene microspheres have been formed by layer-by-layer self-assembly via electrostatic interactions. As a model antigen, rabbit IgG has been bound to the outermost layer of the fluorescent microspheres. The immunoreaction between fluorescent microspheres/rabbit IgG and the corresponding antibody was confirmed by change of the fluorescence spectrum and competitive immunoassay. This approach allowed detection of the antigen (rabbit IgG) in the range 1-500 mg/L, based on the change in the fluorescence intensity of the reporter (fluorescent microspheres/rabbit IgG). A novel microfluidic chip device with a laser-induced fluorescence system was established and used for the detection of fluorescent microspheres in this study.     

Metabolism of testosterone sulfate in the rat: analysis of biliary metabolites.

Following intraperitoneal injection of a mixture of testosterone-7-3-H-17-sulfate and testosterone-4-14-C into male and female rats with bile fistulas, biliary metabolites were separated and purified by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the bile. The major portion of the 3H was excreted in the disulfate fraction in both sexes. Solvolysis of the disulfate revealed the sex-specific aglycone pattern: 5alpha-Androstane-3beta,17beta-diol was the major metabolite in the male rat, whereas 5alpha-androstane-3alpha,17beta-diol and polar steroids were found in the female. In marked contrast, testosterone was metabolized in a different way than testosterone sulfate. 14-C radioactivity was distributed in monoglucosiduronate, monosulfate, and diconjugate fractions. Analysis of the aglycones showed that polar steroids were the main metabolites in the male. In the female, testosterone was metabolized to polar steroids, androsterone, and 5alpha-androstane-3alpha,17beta-diol.     

Characterization of in vivo biotransformations for trastuzumab emtansine by high-resolution accurate-mass mass spectrometry

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) designed for the treatment of HER2-positive cancers. T-DM1 is composed of the humanized monoclonal antibody trastuzumab connected to a maytansine derivative cytotoxic drug, via a nonreducible thioether linker at random lysine residues, and therefore has a very complex molecular structure. It was anticipated that T-DM1 undergoes biotransformations in circulation. However, there was limited knowledge on these structural changes due to bioanalytical challenges. Here, we have investigated the in vivo biotransformations of T-DM1 using a high-resolution accurate-mass (HR/AM) mass spectrometry approach. Three types of biotransformations were characterized for T-DM1 in circulation in tumor-bearing mice, including cysteine or glutathione adduct formation via maleimide exchange, loss of maytansinol via ester hydrolysis, as well as addition of H₂O via linker-drug hydrolysis. These results provide new insights into in vivo catabolism of T-DM1.     

Activation of a new plasma enzyme by antigen-antibody reaction.

The change of enzyme activity in immunized rabbit plasma after addition of the homologous antigen was examined. The activities of N alpha-tosyl-L-arginine methyl ester (TAMe) and N alpha-tosyl-L-lysine methyl ester (TLMe) hydrolysis increased about 15 to 18 days after immunization. This increase was especially marked before the maximal rise of antibody content, and is thought to be related to the IgM antibody not to the IgG antibody. Enzyme activation was strongly inhibited by chelation of Ca2+ with 5 mM disodium ethylenediamine tetraacetate (EDTA), but not by other protease inhibitors, such as epsilon amino-caproic acid (epsilon-ACA), bovine lung kallikrein inhibitor (Trasylol) or soybean trypsin inhibitor (SBTI).     

Lanthanide chelates as new fluorochrome labels for cytochemistry

Anti-rabbit IgG labeled with a new fluorescent europium chelate was used to localize rabbit IgG to human smooth muscle myosin in a histological section. The antibody labeled with the europium chelate could be viewed with a conventional fluorescence microscope with a steady-state light source. This result encourages the development of a time-resolved fluorescence microscope, because a significant improvement in the signal-to-noise ratio can be anticipated.     

Microbial Esterase Detection with Ultraviolet Fluorescence

A method is presented to identify esterase-synthesizing microbial colonies within mixed culture plates by utilizing induced esterase hydrolysis of nonfluorescent butyryl ester of 7-hydroxy-4-methylcoumarin to the highly fluorescent 7-hydroxy-4 methyl umbelliferone. Microscopy procedures for making esterase loci of fungal mycelia visible with this reaction are described.     

Selective cytotoxicity of an antibody-ricin A chain conjugate for human tumor cells

The toxic subunit of a plant ricin has been conjugate by a disulfide bond to a polyclonal rabbit antibody specific for the L-chain of human IgG. Both the antibody and ricin A-chain retained their original biological activity after conjugation. This conjugate proved to be a potent cytotoxin for surface Ig positive Burkitt lymphoma EB-3 cells, growing in vitro and produced 50% inhibition of protein synthesis at level of 1.4 x 10(-9) M. When tested for cytotoxic action on target cells, the composite conjugate molecule was at least 100 times more effective than antibodies alone, ricin A-chain alone or a conjugate ricin A-chain--normal rabbit IgG.     

Electrophoresis '82 : Advanced methods. Biochemical & clinical applications. Proceedings of the International Conference on Electrophoresis,Athens, April 1982 Edited by D. Stathakos Walter de Gruyter; Berlin, 1983 xvi + 867 pages. DM 260.00

A competitive solid-phase immunoassay for the determination of testosterone in serum samples using time-resolved fluorescence is described. The solid phase is a testosterone-3-(O-carboxymethyl)-oxime-ovalbumin conjugate coated to polystyrene microtiter strips. Europium-labelled polyclonal and monoclonal antibodies against testosterone-3-(O-carboxymethyl)-oxime-bovine serum albumin were compared. Their behavior was quite similar although the polyclonal antibody was more sensitive, giving a detection limit of 15 fmol testosterone per assay. Correlation with RIA was very good (r = 0.982 and y = ?0.150 + 0.969x).     

Improving the sensitivity and dynamic range of reagentless fluorescent immunosensors by knowledge-based design

The variable fragment (Fv) of an antibody can be transformed into a reagentless fluorescent biosensor by mutating a residue into a cysteine in the neighborhood of the paratope (antigen-binding site) and then coupling an environment-sensitive fluorophore, e.g., N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD ester), to the mutant cysteine. For some residues, named operational, the formation of the conjugate does not affect the affinity of the Fv fragment for the antigen, and the binding of the antigen generates a measurable variation in the fluorescence intensity of the conjugate. We tested if this signal variation could be increased by coupling several molecules of fluorophores to the same molecule of Fv. Seven operational residues have been previously identified in the single-chain Fv (scFv) of monoclonal antibody D1.3 (mAbD1.3), directed against lysozyme. Ten double mutants of scFvD1.3, involving these residues, were constructed and coupled to the IANBD ester. The fluorescence of the double conjugates revealed a transfer of resonance energy between the two identical fluorescent groups. This homotranfer could be more important in the free state of the conjugate than in its antigen-bound state and increase its sensitivity for the detection of the antigen by up to 2.9-fold. A poorly sensitive conjugate could be improved by coupling a second molecule of fluorophore to residues located far from the paratope. Mutations altering the affinity of scFvD1.3 for lysozyme were introduced into one of its fluorescent conjugates. Using a mixture of three mutant derivatives of this unique conjugate, we could titrate lysozyme with precision in a concentration range encompassing 3 orders of magnitude.     

Magnetic protein microbead-aided indirect fluoroimmunoassay for the determination of canine virus specific antibodies

Rabies, canine distemper, and canine parvovirus are common contagious viral diseases of dogs and many other carnivores, and pose a severe threat to the population dynamics of wild carnivores, as well as endangering carnivore conservation. However, clinical diagnosis of these diseases, especially canine distemper and canine parvovirus, is difficult because of the broad spectrum of symptoms that may be confused with other respiratory and enteric diseases of dogs. The most frequently used and proven techniques for diagnosing viral diseases include the conventional enzyme-linked immunosorbent assay (ELISA), rapid fluorescent focus inhibition test (RFFIT), mouse neutralisation test (MNT), and fluorescent antibody virus neutralization (FAVN) test. However, these methods still have some inherent limitations. In this study, a magnetic protein microbead-aided indirect fluoroimmunoassay was developed to detect canine virus specific antibodies, human rabies immunoglobulin, CDV McAbs, and CPV McAbs. In this assay, an avidin-biotin system was employed to combine magnetic microbeads and virus antigens (rabies virus, canine distemper virus, and canine parvovirus). Quantification of the targeted virus antibodies was analyzed through indirect fluoroimmunoassay using the specific antigen-antibody reaction, as well as their corresponding FITC-labeled detection antibodies (mouse anti-human IgG/FITC conjugate or rabbit anti-dog IgG/FITC conjugate). The results indicated that the fluorescence intensity increased when a higher concentration of the targeted analyte was used, but the control had almost no fluorescence, much like the conventional ELISA. For human rabies immunoglobulin, CDV McAbs, and CPV McAbs, the minimum detectable concentrations were 0.2 IU/mL, 0.3 ng/mL, and 0.5 ng/mL, respectively. All of these results indicate that this assay can be employed to determine the presence of canine virus specific antibodies. In addition, the method devised here can be utilized as a general protocol in other bacterial and viral marker analysis.     

Control of nonspecific staining in the fluorescent antibody technique for the detection of salmonellae in foods.

A fluorescent antibody conjugate, prepared from the IgG (immunoglobulin G) fraction of Salmonella polyvalent flagellar antiserum, gave better specific staining intensities and significantly lower nonspecific staining than did conjugates prepared from globulin fractions of ammonium sulfate-fractionated Salmonella polyvalent antisera. IgG was purified by affinity chromatography against protein A, a normal cell wall component of Staphylococcus aureus. Affinity chromatography yielded high-purity IgG in a one-step purification procedure. The conjugate prepared from affinity-purified IgG was compared with commercially available fluorescent antibody conjugates for the detection of salmoneallae in retail samplings of meats and poultry and gave better correlations with the cultural method than did the commercial conjugates.     

Control of nonspecific staining in the fluorescent antibody technique for the detection of salmonellae in foods.

A fluorescent antibody conjugate, prepared from the IgG (immunoglobulin G) fraction of Salmonella polyvalent flagellar antiserum, gave better specific staining intensities and significantly lower nonspecific staining than did conjugates prepared from globulin fractions of ammonium sulfate-fractionated Salmonella polyvalent antisera. IgG was purified by affinity chromatography against protein A, a normal cell wall component of Staphylococcus aureus. Affinity chromatography yielded high-purity IgG in a one-step purification procedure. The conjugate prepared from affinity-purified IgG was compared with commercially available fluorescent antibody conjugates for the detection of salmoneallae in retail samplings of meats and poultry and gave better correlations with the cultural method than did the commercial conjugates.     

Influence of different combinations of antibodies and penicillinase-labeled testosterone derivatives on sensitivity and specificity of immunoassays.

Three antisera raised against bovine serum albumin (BSA) conjugates of testosterone-3-(O-carboxy-methyl)-oxime (T-3-CMO), 11 beta-hydroxytestosterone-11-carboxymethyl ether (T-11 beta-O-CME) and 19-hydroxytestosterone-19-carboxymethyl-ether (T-19-O-CME) were evaluated in enzyme immunoassays (EIAs) in combinations with penicillinase-labeled T-3-CMO, T-11 beta-O-CME, T-19-O-CME, and testosterone-17 beta-hemisuccinate (T-17 beta-HS) for their influence on the sensitivity and specificity of EIAs. Of the various combinations, anti-T-3-CMO antiserum along with T-11 beta-O-CME-penicillinase showed no cross-reaction with any of the closely related steroids, although the same antibody had 21.6% binding to 5 alpha-dihydrotestosterone (5 alpha-DHT) in radioimmunoassay. All the homologous combinations appeared to be less sensitive due to their low affinity for testosterone. It was also apparent that of all the heterologous systems tested, only two combinations, (a) anti-T-19-O-CME antiserum and T-3-CMO-penicillinase and (b) anti-T-3-CMO antiserum and T-11 beta-O-CME-penicillinase, were found to be more sensitive. The former was less specific; it showed 70% cross-reaction with 5 alpha-DHT. The ability of testosterone to displace the hapten-enzyme conjugate and the specificity of the assay appear to depend on the position of the enzyme label on the steroid molecule as well as on the availability of antigenic sites in particular combinations of antibody and hapten-enzyme conjugates.     

Protection of cultured endothelial cells from hydrogen peroxide-induced injury by antibody-conjugated catalase

The cytoprotective features of catalase-antibody conjugate prepared by covalent conjugation of catalase to rabbit antibody against mouse IgG is described. The bifunctional cross-linking agent m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) was used for conjugation. Functionally active conjugate binds specifically to the plastic-adsorbed mouse IgG and to the surface of live human endothelial cells treated with mouse antiserum against human endothelial cells. Up to 4 units of catalase activity can bind to 1 cm2 of the endothelial monolayer. The targeted catalase protects endothelial cells from cytotoxic action of hydrogen peroxide: the minimal cytotoxic concentration of H2O2 for protected cells is 80-times higher than for intact cells. This effect is attributed partly to local reduction of H2O2 concentration in the cell microenvironment. Targeted catalase was estimated to reduce H2O2 concentration 8-fold near the cell surface with respect to average total concentration.     

Synthesis of site-specific methotrexate-IgG conjugates

Summary With a view to increasing drug incorporation without loss of antibody activity, tritium-labeled methotrexate (MTX) was covalently linked to a polyclonal rabbit IgG antibody against bovine serum albumin and a monoclonal mouse IgG antibody against human renal cancer (Dal K20) by a site-specific method based on hydrazone bond formation between MTX hydrazide and the aldehyde groups generated by periodate oxidation of carbohydrate moieties in IgG (which are uncommon in the antigen-binding region). These conjugates were compared with the corresponding non-site-specific MTX-IgG conjugates produced by the N-hydroxysuccinimide active-ester method with regard to synthesis, stability, retention of antibody activity, inhibition of the target enzyme dihydrofolate reductase and antitumor effect. Incorporation levels achieved with the hydrazide method were no greater than with the active-ester method, typically 6–7 mol MTX/mol IgG. Approximately the same dihydrofolate-reductase-inhibitory capacity was observed for MTX bound by either method. Hydrazide conjugates lost bound drug more rapidly than active-ester conjugates on freezing and thawing, on incubation at 37° C and 51° C, and in the presence of serum or rat liver homogenates. Exposure to rat liver homogenates at 37° C, pH 4.6, for 24 h led to the loss of 50%–60% of the bound drug from hydrazide conjugates compared to 20%–30% from the active ester conjugates. Bio-Gel P-2 chromatography of low-molecular-mass fractions, obtained after exposure of each of the conjugates to liver homogenates, revealed the presence of a compound that had the same elution volume and R _F on thin-layer chromatography as free MTX. Enzyme-linked immunosorbent assay showed loss of antibody activity of both types of conjugates at 51° C and on freezing and thawing. In a clonogenic assay, the active-ester conjugate of Dal K20 appeared to be equally effective or slightly better as a tumor inhibitor than the corresponding hydrazide conjugate. The hydrazide method may be useful in linking MTX to those monoclonal antibodies that tend to denature when subjected to the active-ester method of linkage. Abbreviations used: aBSA, rabbit anti-(bovine serum albumin) IgG; EDCI, 3-ethyl-1-(3-dimethylaminopropyl)carbodiimide; ELISA, enzyme-linked immunosorbant assay; IC₅₀, concentration giving 50% inhibition; MTX, methotrexate; MTXAE, N-hydroxy-succinimide-based active ester of MTX; MTXAE-IgG, MTX-IgG conjugate prepared by the active-ester method; MTXH, methotrexate hydrazide; MTXH-IgG, MTX-IgG conjugate prepared by the hydrazide method; PBS, 0.01 M sodium phosphate, pH 7.1, containing 0.145 M sodium chloride; TLC, thin-layer chromatography     

Selective immunoprecipitate identification using monoclonal anti-rabbit IgG

A monoclonal mouse antibody directed against rabbit IgG has been conjugated with horseradish peroxidase and used to identify immunoprecipitates which contain rabbit antibodies. By combining a specific rabbit antisera with a general antiserum from another species (e.g., goat antiserum against human serum), immunoprecipitates containing the antigen(s) recognized by the rabbit antibodies have been selectively identified by colorimetric development of peroxidase activity. Since the monoclonal antibody is specific for rabbit IgG and nonprecipitating, the peroxidase conjugate can be included in the agarose with the primary antisera.     

Localization of vitellogenin and serum albumin in hepatic parenchymal cells of normal and estradiol-treated immature chickens

The localization of albumin and vitellogenin was determined in liver sections from control and estradiol-treated chickens by two different immunocytochemical techniques: (1) The sandwich technique with rabbit anti-lipovitellin or rabbit anti-albumin IgG and fluorescent goat anti-rabbit IgG and (2) the mixed aggregation immunocytochemical technique with anti-lipovitellin IgG and fluorescent lipovitellin.The results show that the antibody against albumin bound only to all liver parenchymal cells. Furthermore, the fluorescence intensity was equally strong in the portal, intermediate and central zones of the lobules.The fluorescent stain for vittelogenin was not above background in livers of control chicks but was far above background in estradiol-treated chicks. As with albumin the fluorescent stain was distributed equally among the parenchymal cells.The results were quantitatively the same 2 and 4 days after estradiol treatment. The relative rates of synthesis and the concentrations of albumin and vitellogenin correlate well with values obtained for tissue sections by immunocytochemical techniques.     

Multi residue screening of intact testosterone esters and boldenone undecylenate in bovine hair using liquid chromatography electrospray tandem mass spectrometry

The abuse of esters of natural androgenic steroids in cattle fattening and sports is hard to control via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. In veterinary control strange findings of 17beta-testosterone and 17alpha-testosterone in urine are often ignored because of the lack of statistically sound reference data of naturally occurring levels. An interesting alternative for inconclusive urine analyses in veterinary control can be provided by the analysis of the administered steroids themselves, i.e. the analysis of intact steroid esters in hair. Unfortunately, the analysis of intact steroid esters is complicated not only by the vulnerability of the esters which precludes alkaline hydrolysis of the hair, but also by the wide polarity range of short and long-chain esters yielding very poor recoveries for either the one or the other. In this study, a multi-steroid esters LC/MS/MS screening method is presented for trace analysis of the synthetic intact esters of 17beta-testosterone and the undecylenate ester of 17beta-boldenone in bovine hair. The method, requiring only 200 mg of pulverised hair, features a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and the use of four deuterium-labelled steroid esters as internal standards covering the wide polarity range of the analytes. In spiked hair samples for most of the analytes the limit of detection and the accuracy using isotope dilution were 2-5 ng/g and 97-105%, respectively. The applicability was demonstrated using hair samples from a controlled experiment in which six bovines were injected intramuscularly with two different doses of two commercial mixtures of testosterone esters, and with two different doses of boldenone undecylenate. Depending on the dose all administered testosterone- and boldenone esters were found to be incorporated in bovine hair following a single intramuscular injection, except testosterone propionate which dose might have been too low.     

An efficient method for labelling antibodies with 111In

Using a new method, rabbit IgG and a monoclonal antibody have been conjugated with the chelating agent DTPA. This was accomplished with reaction conditions that should entail lower antibody damage than existing methods. Gel filtration of the 111In-labelled antibody conjugate indicated minimal damage to the antibody and radioimmunoassay showed no significant change in its immunological activity.