Characteristics of anti-testosterone antisera produced by bovine serum albumin conjugates of 15 alpha- and 15 beta-carboxymethyltestosterone: use of [125I]iodinated tracers

Each testosterone [125I]iodinated histamine derivative where [125I]iodinated histamines were linked to respective 15 alpha- and 15 beta-carboxymethyltestosterone (15 alpha- and 15 beta-CMT), testosterone-3-(O-carboxymethyl)oxime (T-3-CMO) and testosterone-17 beta-hemisuccinate (T-17-HS) were tested for their usefulness as radiotracers in testosterone immunoassay. In the use of anti-15 alpha- and 15 beta-CMT antisera produced in rabbits against 15 alpha- and 15 beta-CMT-bovine serum albumin (BSA) conjugates, the antisera with 15 alpha- and 15 beta-CMT-[125I]iodinated tracers showed low sensitivity and somewhat low specificity in comparison with those of the antisera with tritiated testosterone (T-3H). On the other hand, the antisera with T-3-CMO-[125I]iodinated tracer showed high sensitivity but low specificity for 5 alpha-dihydrotestosterone (5 alpha-DHT) in comparison with T-3H. The T-17-HS-[125I]iodinated tracer was not bound to the antisera. In the use of anti-15 alpha- and 15 beta-CMT antisera produced in rabbits by pretreatment with 15 alpha-carboxymethyl-5 alpha-DHT linked to a copolymer of D-glutamic acid and D-lysine followed by immunization with 15 alpha- and 15 beta-CMT-BSA, the antisera with homologous [125I]iodinated tracer showed high sensitivity and specificity.     

Preparation and comparative properties of antisera against glyco-3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid linked to bovine serum albumin at the C-3, C-15 alpha, and C-24 positions

Anti glyco-3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid antisera were prepared by immunizing rabbits with hapten-bovine serum albumin (BSA) conjugates coupled at the C-3, C-15 alpha, and C-24 positions on the bile acid molecule, and their properties were investigated by heterologous combination assay using 125I-labeled tracer. The antiserum raised against the C-3 BSA conjugate showed poor titer and specificity, while the antisera from the other two conjugates showed satisfactorily high affinity constants (Ka = 5.0 x 10(8) and 7.0 x 10(8) M-1) and reasonable specificity, exhibiting negligible cross-reactivities with other major human bile acids and cholesterol. Among the unsaturated bile acids tested, high reactivity was observed with tauro-3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid, which suggested that bridge phenomena were significant in this assay system.     

Isolation of flavonoids from Populus nigra as delta 4-3-ketosteroid (5 alpha) reductase inhibitors

Inhibitors of delta 4-3-ketosteroid (5 alpha) reductase, which had been prepared from rat prostate and converted testosterone to 5 alpha-dihydrotestosterone and 4-androstene-3,17-dione, were isolated from 50% ethanol extract of Populus nigra. They were identified as pinobanksin (I, 3,5,7-trihydroxyflavanone), 3,7-dimethylquercetin (II, 3',4',5-trihydroxy-3,7-dimethoxyflavone) and pinocembrin (III, 5,7-dihydroxyflavanone). Compound III showed the most potent inhibitory activity among them.     

Determination method for steroid 5alpha-reductase activity using liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry.

A determination method for steroid 5alpha-reductase activity using liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) in the positive-ion mode has been developed. The rat prostatic enzyme source was used and the enzymatically formed 5alpha-dihydrotestosterone and 5a-androstane-3alpha,17beta-diol were determined by LC/APCI-MS using absolute calibration curve method. The sum of the formed products was used as a measurement of the enzyme activity. This method was applied to kinetic study of this enzyme and inhibitory experiments using Finasteride as a model inhibitor.     

Blue chitin columns for the extraction of heterocyclic amines from cooked meat

The identification of the in vitro metabolites of dehydroepiandrosterone formed from human prostate homogenate was investigated by hyphenated techniques using the stable-isotope dilution method. A mixture of dehydroepiandrosterone and [2H4]dehydroepiandrosterone was incubated with hypertrophied human prostate tissue homogenate in the presence of NAD, NADH and NADPH. The metabolites were extracted with AcOEt-hexane, purified by solid-phase extraction, and then analyzed by LC-atmospheric pressure chemical ionization MS and/or GC-MS. Androst-5-ene-3beta,17beta-diol (major product), androst-4-ene-3,17-dione, testosterone, 5alpha-dihydrotestosterone, androsterone, and 7alpha-hydroxydehydroepiandrosterone were identified in comparison with authentic samples based on their chromatographic behavior and mass spectra.     

Preparation of highly specific anti-zearalenone antibodies by using the cationic protein conjugate and development of an indirect competitive enzyme-linked immunosorbent assay

Although anti-zearalenone (ZEN) antibodies have been widely prepared, these antibodies cross-react with α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL). To overcome this problem and improve the specificity of immunoassays, we produced anti-ZEN antibodies based on a ZEN-cationic protein conjugate. In this study, ZEN was coupled with cationic bovine serum albumin (cBSA) via a Mannich reaction. After BALB/c mice were immunized with ZEN-cBSA, an immunological response was rapidly induced. The titers of the polyclonal antisera and monoclonal antibody were 30,000 and 20,000, respectively. Cross-reactivity (CR) values of the anti-ZEN polyclonal antisera and monoclonal antibody with the 5 analogs were <7% and <2%, respectively. An indirect competitive enzyme-linked immunosorbent assay based on the monoclonal anti-ZEN antibody was established. The recovery rates of ZEN in spiked cereal and feed were in the range of 80%-120% with coefficients of variation <15%. The intra-assay variation and inter-assay variation in assay buffer were both <5%. Therefore, the results demonstrated a feasible approach for preparing highly specific, higher titer and more rapidly induced antibodies against ZEN by using a ZEN-cBSA conjugate as the immunogen instead of currently used immunogens.     

Radioimmunoassay for individual lactate dehydrogenase isoenzymes 1 and 2

Radioimmunoassays (RIA) for the determination of the individual lactate dehydrogenase (LDH) isoenzymes, LDH-1 and LDH-2 have been developed. LDH-1 can be measured in the range of 5–100 ng and LDH-2 in the range of 5–80 ng, if there is no significant cross reactivity. Immunization of several rabbits with LDH-1 and LDH-2 isoenzymes reveals that some animals do not produce antisera to LDH-2 while those injected with LDH-1 generated antiserum in each case. The results of the binding studies suggest that a 50% binding that is recommended for RIA can be achieved with a titer value of 12000 dilution of the antisera. Cross reactivity studies indicate that LDH-1 cross reacts with the antisera to LDH-2 if its concentration is higher than 30 ng/ml of the RIA mixture while LDH-2 cross reacts with the antisera to LDH-1 only if its concentration exceeds 80 ng/ml.     

A radioimmuno-chromatographic scanning method for the analysis of testosterone conjugates in urine and serum

A method for the analysis of testosterone (and 5alpha-dihydrotestosterone) conjugates in human serum and urine samples is described. The samples were grought to pH 1 and extracted with a diethyl ether-methanol mixture. After evaporation the residues were run in a thin-layer chromatography system, individual samples' paths were cut into 1-cm long pieces and eluted with methanol. The methanol was evaporated and the residue subjected to acid hydrolysis. The released steroid was extracted by diethyl ether and measured by radioimmunoassay. The methodology described represents a new approach to the qualitative and quantitative study of steroid conjugates in serum and urine, and can easily be applied to the study of steroid conjugates in other biological mateiral.     

New tools for molecular imaging of redox metabolism: development of a fluorogenic probe for 3 alpha-hydroxysteroid dehydrogenases

A new fluorogenic substrate was developed for 3alpha-hydroxysteroid dehydrogenases (3alpha-HSD), including the human enzymes implicated in important physiological functions (androgen deactivation, neurosteroid activation). While ketone 5 is nonfluorescent, the corresponding alcohol exhibits high fluorescence with emission maximum at 510 nm, thus constituting a redox optical switch. This study began with a chemical concept of a ketone-alcohol optical switch which guided the synthesis of a focused array of compounds. Subsequently, seven compounds were selected (1-7) on the basis of their optical and chemical (stability) properties and were submitted to a screen against a panel of dehydrogenase enzymes. Probe 5 was found to be highly selective for bacterial, rat, and human 3alpha-HSD enzymes. The kinetic parameters were obtained for human 3alpha-HSD enzyme (type 2 isozyme, AKR 1C3; Km = 2.5 muM, kcat = 8.2 min-1). Remarkably, comparison to 5alpha-dihydrotestosterone (5alpha-DHT, Km = 26 muM, kcat = 0.25 min-1, Figure 4), a likely physiological substrate in prostate, revealed that synthetic probe 5 is in fact a far better substrate for this enzyme. Structure 5 represents an exciting lead for the development of a redox imaging probe.     

Heterodimeric Bis(amino thiol) Complexes of Oxorhenium(V) That Mimic the Structure of Steroid Hormones. Synthesis and Stereochemical Issues

We have prepared a series of bis-bidentate complexes of rhenium that mimic the size, shape, and peripheral functionality of steroidal androgens. In a model system, we used 2D NMR and X-ray crystallographic analysis to show that adjacent N-methyl and oxo substitutents adopt an anti configuration during the coordination reaction. We have synthesized a bis-bidentate oxorhenium(V) complex whose structure and peripheral functionality mimic 5alpha-dihydrotestosterone. 2D-NMR analysis indicates that the N-methyl and oxo substituents are driven into the steroidal anti configuration (beta-N-methyl, alpha-oxo) by the beta-orientation of the methyl group equivalent to C-18. Thus, this metal complex provides a remarkable structural and stereochemical mimic of a steroid. Its in vivo stability, however, appears to be limited.     

Determination of plasma testosterone by mass fragmentography using testosterone-19-d3 as an internal standard. Comparison with radioimmunoassay

Analytical procedures for the measurement of testosterone by mass fragmentography (MF) using trideuterated testosterone (testosterone-19,19,19-d3) are described. For the calculation of plasma testosterone, peak height ratios were measured by MF performed on the molecular ions of the TFA derivative of testosterone (m/e 480) and testosterone-19,19,19-d3 (m/e 483). The sensitivity of the method was judged from the lower limit of detection of the mass spectrometer which was at 10 pg. For the measurement of the precision, the inter- and intra-assay coefficients of variation (C.V.) were calculated by using a pooled plasma sample; they were 3.15% and 1.79%, respectively. The specificity was investigated by the use of 5 alpha-dihydrotestosterone and the MF method was found to afford a highly selective technique. These results obtained by MF have been compared with the results obtained by a radioimmunoassay method.     

Studies on neurosteroids XX. Liquid chromatography-tandem mass spectrometric method for simultaneous determination of testosterone and 5alpha-dihydrotestosterone in rat brain and serum

Testosterone (T) and 5alpha-dihydrotestosterone (DHT) are now referred to not only as androgenic steroid hormones, but also as neuroactive steroids, because they elicit anesthetic and anxiolytic effects. Methods using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) coupled with derivatization are developed and validated to examine rat brain and serum levels of T and DHT and their stress-induced changes. The steroids are extracted with methanol-acetic acid from the brain tissue or serum, purified using solid-phase extraction cartridges, derivatized with a permanently charged reagent, 2-hydrazino-1-methylpyridine, and subjected to LC-MS-MS. [19,19,19-(2)H3]-T is used as the internal standard. The intra- and inter-assay coefficients of variation are below 10.0%, and the analytical recoveries are 98.1-103.0%. The developed methods are applied to the animal study and it was found that a fair amount of DHT is continuously and locally synthesized in the brain, and its level is not changed by the immobilization stress and depends on the brain T level.     

Conjugated 1 beta-hydroxycholic acid in the urine of newborns and pregnant women measured by radioimmunoassay using antisera raised against N-(1 beta-hydroxycholyl)-2-aminopropionic acid-bovine serum albumin conjugate.

Anti-tauro 1 beta-hydroxycholic acid antisera were prepared by immunizing rabbits with N-(1 beta,3 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholan-24-oyl)-2- aminopropionic acid-bovine serum albumin (BSA) conjugate. The antisera raised had high affinity (1.25-1.46 x 10(9) M-1) and specificity for conjugated 1 beta-hydroxycholic acid; cross-reactivity for glyco 1 beta-hydroxycholic acid was 100% and that for the glycine and taurine conjugates of other 1 beta-hydroxylated bile acids ranged from 11.30 to 0.23%. Urinary concentrations of conjugated 1 beta-hydroxycholic acid were determined by radioimmunoassay in newborns, 0-20 d after birth, in amounts ranging from 0.29 to 18.51 micrograms/ml and in women in late pregnancy (less than 1.13 micrograms/ml), as well as in normal women (less than 0.12 microgram/ml).     

Assay methods for 6-keto-prostaglandin F1 alpha in human urine. Comparison of chromatographic techniques with radioimmunoassay and gas chromatography-negative-ion chemical-ionization mass spectrometry

6-Keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) in human urine is considered to be a reflection of renal prostacyclin production. Because of the large amounts of unidentified eicosanoid metabolites in urine that may potentially bind to 6-keto-PGF1 alpha antisera, most radioimmunoassays include chromatographic purification of urine. A comparison of chromatographic techniques and of antisera to 6-keto-PGF1 alpha for the assay of human urine is described. Gas chromatography--negative-ion chemical-ionization mass spectrometry (GC--NICI-MS) was used as the reference method. Radioimmunoassays were performed with each of four antisera combined with each of three chromatographic purification systems (silicic acid, Sephadex LH-20, reversed-phase high-performance liquid chromatography). There was marked variability in the results; however, there was at least one chromatographic method for each antiserum that yielded results comparable to GC--NICI-MS. Direct radioimmunoassay of urine without chromatography yielded markedly elevated and variable results for the four antisera. In contrast, the four antisera gave very similar results with direct assay of media from isolated perfused organs. Thus, for the radioimmunoassay of 6-keto-PGF1 alpha in human urine, each antiserum is sensitive to different contaminants in urine and must be individually matched to a chromatographic purification system.     

Characterization of Anti-Chloramphenicol Antibodies by Enzyme-Linked Immunosorbent Assay

Polyclonal antibodies against conjugates of chloramphenicol succinate and chloramphenicol base with proteins were obtained and characterized in direct ELISA. Antiserum against a conjugate of chloramphenicol (CAP) base with BSA (direct coupling) was very specific and showed cross-reactivity only with CAP succinate (11.3%) and CAP base (4.6%); whereas, antisera against a conjugate of CAP succinate with a protein recognized CAP succinate strongly as an initial compound. In direct ELISA, antisera against a conjugate of CAP succinate with KLH (homologous assay) and CAP base with BSA (heterologous assay) showed similar sensitivity: IC₅₀ were 1.3 and 1.5 ng mL^?1, respectively. Applicability of the immunoreagents obtained was shown in the analysis of CAP residues in milk (3.5% fat content). Detection limit of 0.3 ng mL^?1 was obtained for milk diluted 5 times.     

Studies on human proinsulin C-peptide radioimmunoassay method--preparation of antiserum and its specificity-- (author's transl)]

The antisera using at final dilution of 1 : 10,000 have been prepared by immunizing synthetic human proinsulin connecting peptide to rabbits for human proinsulin C-peptide radioimmunoassay. The cross reactivities of human proinsulin C-peptide derivatives with the prepared antisera were reduced by leaving amino acid residues from N terminal, although this phenomenon was a little different among antisera. Those results suggested that main antigen determinant in N terminal 31-38 of human proinsulin connecting peptide. The cross reactivities of other animal proinsulin C-peptide and other peptide hormones with the prepared antissera were not recognized at 10(3) p mole/ml.     

Production and specificity of anti-22-oxacalcitriol antisera.

22-Oxacalcitriol 3-hemiglutarate, a haptenic derivative of 22-oxacalcitriol, was synthesized to obtain a specific antibody for use in radioimmunoassay. Three antisera were elicited in rabbits against the hapten conjugated with bovine serum albumin, and their specificity was examined by cross-reaction study. One of the antisera was found to be satisfactorily specific, and expected to provide a radioimmunoassay useful for the pharmacokinetic study of 22-oxacalcitriol.     

The accessibility of antigenic determinants of ribosomal protein S4 in situ.

Antibodies to Escherichia coli ribosomal protein S4 react with S4 in subribosomal particles, eg, the complex of 16S RNA with S4, S7, S8, S15, S16, S17, and S19 and the RI reconstitution intermediate, but they do not react with intact 30S subunits. Antibodies were isolated by three different methods from antisera obtained during the immunization of eight rabbits. Some of these antibody preparations, which contained contaminant antibodies directed against other ribosomal proteins, reacted with subunits, but this reaction was not affected by removal of the anti-S4 antibody population. Other antibody preparations did not react with subunits. It is concluded that the antigenic determinants of S4 are accessible in some protein deficient subribosomal particles but not in intact 30S subunits.     

Characterisation of antibodies to chloramphenicol, produced in different species by enzyme-linked immunosorbent assay and biosensor technologies

Six polyclonal antisera to chloramphenicol (CAP) were successfully raised in camels, donkeys and goats. As a comparison of sensitivity, IC₅₀ values ranged from 0.3 ng mL⁻¹ to 5.5 ng mL⁻¹ by enzyme-linked immunosorbent assay (ELISA) and from 0.7 ng mL⁻¹ to 1.7 ng mL⁻¹ by biosensor assay. The introduction of bovine milk extract improved the sensitivity of four of the antisera by ELISA and two by biosensor assay; a reduction in sensitivity of the remaining antisera ranged by a factor of 1.1-2.6. Porcine kidney extract reduced the sensitivity of all the antisera by a factor ranging from 1.1 to 7 by ELISA and a factor of 1.5 to 4 by biosensor. A low cross-reactivity with thiamphenicol (TAP) and florfenicol (FF) was displayed by antiserum G2 (1.2% and 18%, respectively) when a homologous ELISA assay format was employed. No cross-reactivity was displayed by any of the antisera when a homologous biosensor assay format was employed. Switching to a heterologous ELISA format prompted three of the antisera to display more significant cross-reactivity with TAP and FF (53% and 82%, respectively, using D1). The heterologous biosensor assay also increased the cross-reactivity of D1 for TAP and FF (56% and 129%, respectively) and of one other antiserum (G1) to a lesser degree. However, unlike the ELISA, the heterologous biosensor assay produced a substantial reduction in sensitivity (by a factor of 6 for D1).