Development of a strategy for biological monitoring in a chemical plant producing 3,3'-dichlorobenzidine dihydrochloride

In a chemical plant in Germany producing 3,3'-dichlorobenzidine dihydrochloride for the manufacture of colorants, blood and urine samples were taken for biological monitoring. 3,3'-Dichlorobenzidine (DBZ) was analyzed in urine by thin-layer chromatography and subsequently further combined with analysis of adducts of 3,3'-DBZ in hemoglobin. Data highlight current ranges of industrial exposure to 3,3'-DBZ in Germany and demonstrate the applicability of biological monitoring to minimize this exposure. Effective biological monitoring was achieved by a combination of monitoring hemoglobin adducts with spot samplings of urinary 3,3'-DBZ excretion in cases of reported exposure periods. Data presented might help to identify biological guidance values (BGV/BAR) for 3,3'-DBZ-exposed individuals.     

A kinetic enzyme immunoassay for the quantitation of antibodies to a humanized monoclonal antibody in human serum

A kinetic enzyme immunoassay was developed and validated to quantitate human antibodies to the humanized monoclonal antibody CAMPATH¹-1H (C1H) in human serum. The assay was configured using C1H-coated 96-well plates which were blocked with bovine serum albumin, and incubated with dilutions of human serum containing anti-C1H antibody. Antibody was detected using biotinylated C1H followed by streptavidin-conjugated alkaline phosphatase and p-nitrophenyl phosphate. Absorbance data were collected for 10 min, and mOD min⁻¹ data were exported to MultiCalc data analysis software. A 4-parameter logistic-log algorithm was shown to model the data through the range of the standard curve within 15% of nominal values. The overall assay performance coefficient of variation by ANOVA was 9.2%. The lower limit of detection was defined at 160 Units ml⁻¹. The anti-idiotype antibody standard stock solution is stable at 4°C and at −80°C for at least 11 months in buffer. The anti-idiotype antibody controls are stable for at least seven freeze–thaw cycles and at least 6 months in human serum stored at −20°C. A strategy was devised by which to establish the specific antibody potency for any given batch of anti-C1H antibody standard relative to the Reference Standard. This EIA has been used to quantify and characterize anti-C1H antibody in human serum in support of clinical safety and efficacy studies.     

Development of an enzyme immunoassay for serum 16-dehydropregnenolone

We have developed an enzyme immunoassay (EIA) for serum 16-dehydropregnenolone (3beta-hydroxy-5,16-pregnadien-20-one; 16-DHP). The antiserum against 16-DHP-3-hemisuccinate conjugated bovine serum albumin (16-DHP-3HS-BSA) was raised in rabbits. For use as an enzyme labeled antigen, 16-DHP-3HS was conjugated to alkaline phosphatase. The minimal amount of 16-DHP detected was 4 pg (0.013 pmol)/assay and the measurable range was from 0.06-60 ng/ml (0.191-191 nmol/l). The intra-assay coefficient of variation (C.V.) was 4.1% (0.73+/-0.03 ng/ml, mean+/-S.D., n=6), and inter-assay C.V. was 7.7% (0.13+/-0.01 ng/ml, n=6). A liner relation was observed between the serum sample dilution and the 16-DHP concentration. For the recovery study, authentic 16-DHP was added to a serum sample (original concentration: 0.10-0.14 ng/ml), and the recovery was found to be 94.4-96.8% (final 16-DHP concentrations calculated: 0.29-16.3 ng/ml). To investigate the reliability of the present EIA, the values from our EIA were compared with those obtained by GC-MS. The 16-DHP concentration could not be measured in serum by GC-MS because of its sensitivity. Therefore, the conjugated steroid, 16-DHPS, was first enzymatically hydrolysed and then the 16-DHP measured by both methods. There was a good correlation between the levels determined by these methods (Pearson's correlation coefficient: r=0.927, p<0.001, y=0.74x+3.61, n=27). The serum concentrations of 16-DHP in neonates and umbilical vein were 0.53+/-0.09 ng/ml and 0.88+/-0.61 ng/ml, respectively. No 16-DHP was detected in serum from normal healthy adults using the present EIA. These results suggest that 16-DHP originates from the fetus and neonate.     

Development of a sensitive enzyme immunoassay for measuring taipan venom in serum

The detection and measurement of snake venom in blood is important for confirming snake identification, determining when sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Venom enzyme immunoassays (EIA) have had persistent problems with poor sensitivity and high background absorbance leading to false positive results. This is particularly problematic with Australasian snakes where small amounts of highly potent venom are injected, resulting in low concentrations being associated with severe clinical effects. We aimed to develop a venom EIA with a limit of detection (LoD) sufficient to accurately distinguish mild envenoming from background absorbance at picogram concentrations of venom in blood. Serum samples were obtained from patients with taipan bites (Oxyuranus spp.) before and after antivenom, and from rats given known venom doses. A sandwich EIA was developed using biotinylated rabbit anti-snake venom antibodies for detection. For low venom concentrations (i.e. <1 ng/mL) the assay was done before and after addition of antivenom to the sample (antivenom difference method). The LoD was 0.15 ng/mL for the standard assay and 0.1 ng/mL for the antivenom difference method. In 11 pre-antivenom samples the median venom concentration was 10 ng/mL (Range: 0.3–3212 ng/mL). In four patients with incomplete venom-induced consumption coagulopathy the median venom concentration was 2.4 ng/mL compared to 30 ng/mL in seven patients with complete venom-induced consumption coagulopathy. No venom was detected in any post-antivenom sample and the median antivenom dose prior to this first post-antivenom sample was 1.5 vials (1–3 vials), including 7 patients administered only 1 vial. In rats the assay distinguished a 3-fold difference in venom dose administered and there was small inter-individual variability. There was small but measurable cross-reactivity with black snake (Pseudechis), tiger snake (Notechis) and rough-scale snake (Tropidechis carinatus) venoms with the assay for low venom concentrations (<1 ng/mL). The use of biotinylation and the antivenom difference method in venom EIA produces a highly sensitive assay that will be useful for determining antivenom dose, forensic and clinical diagnosis.     

Comparison of a fluorescent immunoassay with an enzyme immunoassay and a radioimmunoassay for gentamicin

A fluorescent immunoassay (FIA) for gentamicin was evaluated and compared with an enzyme multiplied immunoassay (EMIT) and a radioimmunoassay (RIA) for gentamicin. Pooled human serum that contained low, medium, and high concentrations of gentamicin sulfate (approximately 2.5, 5, and 10 micrograms/ml of gentamicin) were analyzed for actual gentamicin concentration by FIA. Samples were assayed 10 times during the same day to evaluate within-run precision and on 10 different days to evaluate between-run precision. Serum samples obtained from patients receiving gentamicin therapy were analyzed for gentamicin concentration using FIA and EMIT, and separate serum samples were analyzed using FIA and RIA. Results were compared by regression analysis. Within-run coefficients of variation were 8.47%, 6.84%, and 2.62%, respectively, for the low, medium, and high concentrations of gentamicin, and the respective between-run coefficients of variation were 10.81%, 6.31%, and 1.64%. The correlation coefficient for the comparison of FIA with EMIT was 0.943, and the correlation coefficient for the comparison of FIA with RIA was 0.970. The fluorescent immunoassay is a reliable method for determining the concentration of gentamicin in serum. Although the results obtained by FIA correlate well with those obtained by EMIT and RIA, variability exists between concentrations determined by each of these methods.     

Performance of a microtiter plate ELISA for screening of postmortem blood for cocaine and metabolites

The object of this study was to evaluate the suitability of the Neogen Corporation microtiter plate enzyme-linked immunoassay (ELISA) for cocaine and metabolites for screening of postmortem blood. Sixty-five postmortem whole blood specimens were obtained from drug-involved deaths that had been screened and confirmed positive for cocaine and/or benzoylecgonine (BE). Fifty-eight negative specimens were obtained from noncocaine-involved deaths. Specimens were tested using the Neogen Cocaine/BE microtiter plate ELISA assay. No matrix effects were found for whole blood in this assay. The effect of dilutions of the whole blood specimens of 1:5 through 1:50 was studied. A dilution of 1:5 was chosen to correspond to that used for other Neogen microtiter plate assays for drugs in whole blood. True positives, true negatives, false positives, and false negatives were determined and graphed for the ELISA results against gas chromatography-mass spectrometry (GC-MS), GC-nitrogen-phosphorus detection, and case histories. From these graphs and the receiver operating characteristic curves, the optimal cutoff for the Neogen Cocaine/BE ELISA was found to be 5 ng/mL BE equivalents at a 1:5 dilution. The optimum cutoff for a 1:50 dilution was 50 ng/mL BE equivalents. The Neogen Cocaine/BE ELISA had a sensitivity of 93.8% +/- 2.9% and a specificity of 96.6% +/- 2.4% versus GC-MS at a cutoff of 5 ng/mL BE equivalents (1:5 dilution) and a sensitivity of 100% +/- 0.5% and specificity of 98.3% +/- 1.7% versus GC-MS at a 50 ng/mL BE equivalents cutoff (1:50 dilution).     

Validation of a microtiter plate ELISA for screening of postmortem blood for opiates and benzodiazepines

The object of this study was to evaluate the suitability of the Neogen Corp. microtiter plate enzyme-linked immunoassays (ELISA) for opiates and benzodiazepines for screening of postmortem blood. Ninety postmortem whole blood specimens were obtained from drug-involved deaths which had been screened and confirmed positive for opiates and/or benzodiazepines. Forty negative specimens were obtained from non-opiate-involved deaths. Specimens were tested using the Neogen Opiates Group and Neogen Benzodiazepines Group microtiter plate ELISA assays. No matrix effects were found for whole blood in these assays and a dilution of 1:5 was chosen to facilitate pipetting and to bring the IC50 of the microtiter plate ELISA assay within the range of opiates and benzodiazepines encountered in medical examiner specimens. True positive, true negatives, false positives, and false negatives were determined and graphed for the ELISA results against gas chromatography-mass spectrometry (GC-MS), gas chromatography-nitrogen-phosphorus detection and case histories. From these graphs and the ROC curves, the optimal cut-off for the Neogen Opiates Group ELISA was found to be between 20 and 50 ng/mL morphine equivalents and the optimum cut-off for the Neogen Benzodiazepines Group ELISA was between 20 and 50 ng/mL temazepam equivalents. The Neogen Opiates Group ELISA had a sensitivity of 95.2% +/- 2.7% and a specificity of 92.2% +/- 3.4% versus GC-MS at a cut-off of 20-ng/mL cut-off and a sensitivity of 88.8% +/- 3.9% and specificity of 96.8% +/- 2.1% versus GC-MS at a 50-ng/mL morphine equivalents cut-off. The Neogen Benzodizepines Group ELISA had a sensitivity of 100% +/- 1.3% and a specificity of 94.6% +/- 2.9% versus GC-MS (20-ng/mL temazepam equivalents cut-off) and a sensitivity of 95.8% +/- 2.5% and specificity of 98.2% +/- 1.8% versus GC-MS at a 50-ng/mL cut-off.     

Covalent interaction of 3,3'-dichlorobenzidine with hepatic lipids. Enzymic basis and stability of the adducts

Administration of a single oral dose (20 mg/kg) of [U-14C]3,3'-dichlorobenzidine to rats resulted in the in vivo covalent binding of the compound to hepatic lipids. More than 70% of the lipid-3,3'-dichlorobenzidine adducts were accounted for in microsomes. Loss of the lipid-bound 3,3'-dichlorobenzidine residues from either total liver or endoplasmic reticulum occurred in at least two phases--an initial fast phase and a terminal slow phase. In vitro studies with hepatic microsomes in the presence of antibodies to specific P450 isozymes and chemical inhibitors to determine the enzymes that activate 3,3'-dichlorobenzidine to the lipid-binding derivative(s) implicated cytochrome P450d. The 3,3'-dichlorobenzidine-bound microsomal lipids were not mutagenic to Salmonella TA98 in the Ames test. The results suggest that adduct formation between 3,3'-dichlorobenzidine and membrane lipids may provide a measure of 3,3'-dichlorobenzidine activation. It is speculated that covalent interaction of the compound with membrane lipids may modify cellular processes, leading to either enhancement or attenuation of carcinogenesis by the chemical.     

Development of a sensitive enzyme immunoassay for the determination of vinpocetine in human plasma.

An Enzyme immunoassay for the quantitative determination of vinpocetine (CAS-42971-09-5) in human plasma has been developed. The lower limit of quantification is 0.1 ng/ml plasma. The assay shows no cross reactivity with the major metabolite apovincaminic acid. Because of a strong unspecific binding of vinpocetine to plasma proteins an extraction step was necessary. The inter- and intra-assay reproducibility of the test (coefficient of variation) is in a range of 1.1 and 18.3%.     

Development of a flow cytometric immunoassay for recombinant bovine somatotropin-induced antibodies in serum of dairy cows

Administration of recombinant bovine somatotropin (rbST) to enhance milk production in dairy cows is banned within the European Union. Therefore, methods for pinpointing rbST abuse are required. Due to the problematic detection of rbST itself in serum, methods are also focused on detecting changes in rbST‐related biomarkers. In this study, a fast and easy‐to‐perform microsphere‐based flow cytometric immunoassay (FCIA) for detection of rbST‐induced antibodies in serum was developed. Until now, detection of rbST‐induced antibodies was also problematic due to non‐specific binding of serum proteins resulting in a high rate of false positive results. Therefore, five different sample preparation methods, i.e. dilution, octanoic acid precipitation, filtration, protein G purification, and a previously described generic FCIA sample preparation were critically compared to overcome non‐specific binding to the microspheres. Only the generic FCIA sample pretreatment was effective in reducing non‐specific binding. As a result, an absolute decision level for detecting rbST antibodies in serum of dairy cows was determined and its applicability was demonstrated. In accordance with biological expectations from literature, rbST antibodies were induced in three out of four rbST‐treated dairy cows. These rbST‐induced antibodies were successfully detected for up to 4 weeks after the last rbST treatment, whereas no false positive results were obtained for 27 untreated dairy cows. This is the first method, able to overcome the interference of serum proteins and therefore, can be applied with high confidence for screening unknown herds of cattle for rbST antibodies, an important biomarker for pinpointing at rbST abuse in cattle. Copyright © 2011 John Wiley & Sons, Ltd.     

Rapid microtiter plate assay for determination of inulin in human plasma and dialysate

A rapid, sensitive, and reproducible microtiter plate assay for the determination of inulin in human plasma, dialysate, and phosphate-buffered saline (PBS) was developed. Plasma or PBS samples (100 microl aliquots) were prepared by the addition of indole-3-acetic acid (150 microl) and HCl (3 ml) and then briefly vortex-mixed. Samples were then incubated in a 60 degrees C water bath for 20 min, cooled in a room temperature water bath for 40 min, then diluted with deionized, distilled water (DDW; 3 ml) and again vortex-mixed. Finally, an aliquot (200 microl) of each sample was transferred to a 96-well microtiter plate and read spectrophotometrically at 490 nm. Dialysate samples were processed in a similar manner, but required an initial enzymatic step in order to remove dextrose and minimize assay interference. Samples (100 microl aliquots) were prepared by the addition of glucose oxidase/catalase solution (100 microl), briefly vortex mixed, and then incubated in a 37 degrees C water bath for 60 min, samples were then reacted with indole-3-acetic acid as before. Calibration curves were linear over the concentration range of 0.5-4 mg/ml or 0.025-0.4 mg/ml for plasma or PBS and dialysate, respectively; correlation coefficients (r(2)) were >0.99. The intra- and inter-day coefficients of variation in plasma, PBS, and dialysate were <15%. This method is well suited for the rapid analysis of large numbers of samples and is currently being used for in vitro investigations of solute removal by hemodialysis.     

Development of generic continuous-flow enzyme immunoassay system for analysis of aminoglycosides in serum

A simple generic continuous-flow enzyme immunoassay (CFEIA) for analysis of aminoglycosides in serum has been successfully developed. The developed assay employed a specific monoclonal antibody and beta-galactosidase (beta-GAL) enzyme as label. The assay involves an off-line competitive binding reaction between the analyte and free labelled analyte for the binding sites of the antibody. After equilibrium is reached, the sample was injected into the flow system. The bound antibody complexes with the analyte and the labelled analyte were trapped in a protein G column, while the unbound free labelled analyte was eluted and detected colorimetrically down-stream, after reaction with chlorophenolic red-beta-D-galactopyranoside as a substrate for the beta-GAL enzyme. The concentration of the analyte in a sample was quantified by its ability to inhibit the binding of the analyte-enzyme conjugate to the antibody, and the signal was directly proportional to the concentration of the analyte in the original sample. The optimum conditions for the developed CFEIA were investigated and applied to the analysis of tobramycin, as a representative example of the aminoglycosides, in serum samples. The detection limit of the assay was 0.06 microgml(-1). The assay showed good precision; the coefficients of variation were 2.49-4.33 and 3.30-6.82% for intra- and inter-assay precision, respectively. Serum matrix constituents and the endogenous compounds did not interfere with the assay. Analytical recovery of spiked tobramycin, in the concentration range between 0.5 and 8.0 microgml(-1), was 101.55+/-3.14. The assay results correlated well with those obtained by high-performance liquid chromatography (r=0.991). All the obtained results strongly demonstrate that the developed CFEIA is a suitable method for a rapid and reliable analysis of aminoglycosides in serum.     

Evaluation of PAF antagonists using human neutrophils in a microtiter plate assay

This paper describes a testing model for the detection and evaluation of PAF antagonists, based on the inhibition of PAF-elicited elastase release by human neutrophils. Incubations are performed in microtiter plates in the presence of a specific fluorogenic elastase substrate allowing direct measurement of the exocytosis response by means of a 96-well fluorescence reader. Determinations of the IC50 values for five established PAF antagonists, Ro 19-3704, BN 52021, CV-3988, 48740 RP and kadsurenone, showed that the new model is comparable in sensitivity and discriminative capacity to other in vitro assays. From the effect of antagonists on the PAF concentration-response curve pA2 values could be calculated and information on the type of antagonism obtained. BN 52021 was found to behave as a competitive antagonist while Ro 19-3704 showed a more complex type of inhibition. As a one-plate system, the test is simple to handle and highly reproducible, and appears therefore particularly useful for large drug screening programs.     

Inhibition of CYP3A4 in a rapid microtiter plate assay using recombinant enzyme and in human liver microsomes using conventional substrates.

Cytochrome P450 inhibition studies are performed in the pharmaceutical industry in the discovery stage to screen candidates that may have the potential for clinical drug-drug interactions. A 96-well microtiter plate assay using recombinant cytochrome P450 (Supersomes) has been used to increase the overall throughput. The IC(50) values for the inhibition of CYP3A4 by 52 new chemical entities (NCEs) were determined using the Supersomes assay with resorufin benzyl ether as a substrate, and the data were compared with those obtained in human liver microsomes (HLM) using midazolam as a substrate. Among the 52 compounds tested, 25 showed IC(50) values within a 5-fold difference in the two assays. For all compounds that showed a >5-fold difference, the IC(50) values in the Supersomes assay were lower than those obtained in HLM, except for one compound. Further studies suggested that this discrepancy was not related to difference in protein concentrations between the two assays. In addition, the IC(50) values for 16 compounds with a wide range of inhibition potency were determined in HLM using testosterone and dextromethorphan as substrates. The results showed an 80 to 93% match within a 5-fold difference between the three probe substrates. However, for certain compounds including ketoconazole, there were substrate-dependent differences in the inhibition. The results suggest that the difference between the Supersomes and HLM could be partially attributed to differences in the substrate used, and to metabolism by other cytochrome P450s present in the HLM but not in the Supersomes. Furthermore, multiple CYP3A4 substrates should be used to improve the reliability of estimating potential drug-drug interaction of NCEs.     

抗美沙酮单克隆抗体制备、鉴定和在侧向免疫层析方法中的应用

目的:制备抗美沙酮(MTD)单克隆抗体(mAb),并将筛选到的抗体应用于侧向免疫层析方法检测尿液中MTD。方法:用美沙酮BSA偶联蛋白(MTD-BSA)免疫纯系Balb/c小鼠,采用直接及间接ELISA法进行药物交叉反应,筛选获得能稳定分泌抗MTD的单克隆杂交瘤细胞株。制备腹水,纯化后得到特异性抗MTD单克隆抗体(mAb),进行效价、特异性、纯度、亚类的鉴定分析,并用筛选到的抗体建立检测MTD的侧向免疫层析方法。结果:成功获得1株稳定分泌抗体的阳性细胞株,用建立的侧向免疫层析方法检测临床样品与对照方法比较总符合率为100%。结论:成功筛选出了能稳定分泌mAb的细胞株,抗体应用于侧向免疫层析检测方法中,能快速、特异、灵敏地检测出临床样品中的MTD,为临床应用快速检测MTD指标提供了方法。