John E. Snawder

Comparison of a Multiplexed Fluorescent Covalent Microsphere Immunoassay and an Enzyme-Linked Immunosorbent Assay for Measurement of Human Immunoglobulin G Antibodies to Anthrax Toxins

ABSTRACT Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum (C. P. Quinn, V. A. Semenova, C. M. Elie et al., Emerg. Infect. Dis. 8:1103-1110, 2002). The ELISA had a minimum detectable concentration (MDC) of 0.06 μg/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 μg of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 μg/ml, while the dynamic range was 0.06 to 1.7 μg/ml. The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax. A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100%. We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for B. anthracis PA which was Luminex xMap technology. The FCMIA MDC was 0.006 μg of anti-PA IgG per ml, the RDL was 0.016 μg/ml, and the whole-serum equivalent MDC was 1.5 μg/ml. The dynamic range was 0.006 to 6.8 μg/ml. Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis. The two methods had a high positive correlation (r2 = 0.852; P < 0.001). The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection.

Covariation of Human Microsomal Protein Per Gram of Liver with Age: Absence of Influence of Operator and Sample Storage May Justify Interlaboratory Data Pooling

Scaling of metabolic clearance values from liver microsomal data or recombinantly expressed cytochrome P450 enzymes to predict human hepatic clearance requires knowledge of the amount of microsomal protein per gram of liver (MPPGL). Identification of physiological covariates of MPPGL requires analysis of values from large diverse populations, which necessitates pooling of data from numerous sources. To ensure compatibility between results obtained within and between studies, the impact of interoperator differences and sample storage on values of MPPGL was investigated. With use of triplicate samples from one liver (HL86), no statistically significant difference was detected between values of MPPGL prepared from samples stored at -80°C (23.5 ± 1.2 mg g-1) and those determined using fresh tissue (21.9 ± 0.3 mg g-1). Although there was a significant difference in the yield of microsomal protein obtained from another liver sample (HL43) by three different operators (17 ± 1, 19 ± 2, and 24 ± 1 mg g-1; p = 0.004, analysis of variance), no difference was observed in the estimated MPPGL after application of appropriate correction factors for each operator (28 ± 1, 30 ± 5, and 31 ± 4 mg g-1). The result provided justification for pooling reported values of MPPGL for use in covariate analysis. Investigation of the relationship between age and MPPGL provided preliminary evidence that MPPGL values increase from birth to a maximum of 40 mg g-1 [95% confidence interval for the geometric mean (95% CI meangeo): 37–43 mg g-1 at approximately 28 years followed by a gradual decrease in older age (mean of 29 mg g-1 at 65 years; 95% CI meangeo: 27–32 mg g-1). Accordingly, appropriate age-adjusted scaling factors should be used in extrapolating in vitro clearance values to clinical studies.

Rapid, Sensitive, and Specific Lateral-Flow Immunochromatographic Device To Measure Anti-Anthrax Protective Antigen Immunoglobulin G in Serum and Whole Blood

ABSTRACT Evidence from animals suggests that anti-anthrax protective antigen (PA) immunoglobulin G (IgG) from vaccination with anthrax vaccine adsorbed (AVA) is protective against Bacillus anthracis infection. Measurement of anti-PA IgG in human sera can be performed using either enzyme-linked immunosorbent assay or fluorescent covalent microsphere immunoassay (ELISA) (R. E. Biagini, D. L. Sammons, J. P. Smith, B. A. MacKenzie, C. A. Striley, V. Semenova, E. Steward-Clark, K. Stamey, A. E. Freeman, C. P. Quinn, and J. E. Snawder, Clin. Diagn. Lab. Immunol. 11:50-55, 2004). Both these methods are laboratory based. We describe the development of a rapid lateral-flow immunochromatographic assay (LFIA) test kit for the measurement of anti-PA IgG in serum or whole-blood samples (30-μl samples) using colloidal gold nanoparticles as the detection reagent and an internal control. Using sera from 19 anthrax AVA vaccinees (anti-PA IgG range, 2.4 to 340 μg/ml) and 10 controls and PA-supplemented whole-blood samples, we demonstrated that the LFIA had a sensitivity of approximately 3 μg/ml anti-PA IgG in serum and ∼14 μg/ml anti-PA IgG in whole blood. Preabsorption of sera with PA yielded negative anti-PA LFIAs. The diagnostic sensitivity and specificity of the assay were 100% using ELISA-measured anti-PA IgG as the standard. This kit has utility in determining anti-PA antibody reactivity in the sera of individuals vaccinated with AVA or individuals with clinical anthrax.

Variance of Microsomal Protein and Cytochrome P450 2E1 and 3A Forms in Adult Human Liver.

Differences in the pharmacokinetics of xenobiotics among humans makes them differentially susceptible to risk. Differences in enzyme content can mediate pharmacokinetic differences. Microsomal protein is often isolated from liver to characterize enzyme content and activity, but no measures exist to extrapolate these data to the intact liver. Measures were developed from up to 60 samples of adult human liver to characterize the content of microsomal protein and cytochrome P450 (CYP) enzymes. Statistical evaluations are necessary to estimate values far from the mean value. Adult human liver contains 52.9 ± 1.476 mg microsomal protein per g; 2587 ± 1.84 pmoles CYP2E1 per g; and 5237 ± 2.214 pmols CYP3A per g (geometric mean ± geometric standard deviation). These values are useful for identifying and testing susceptibility as a function of enzyme content when used to extrapolate in vitro rates of chemical metabolism for input to physiologically based pharmacokinetic models which can then be exercised to quantify the effect of variance in enzyme expression on risk-relevant pharmacokinetic outcomes.

Susceptibility to the ototoxic properties of toluene is species specific

Toluene is the most widely used industrial solvent. It has been shown to be ototoxic in mice and rats, and to increase permanent threshold shift in conjunction with exposure to noise. Chinchillas are widely used for studying noise effects on the cochlea. The present study was initiated to study toluene and noise interaction in chinchillas. Thirty-three chinchillas were exposed to a 95 dBA 500 Hz octave band noise plus 2000 ppm toluene, 8 or 12 h per day for 10 days. Auditory function was estimated using the auditory brainstem response (ABR) to tones between 500 Hz and 16 kHz. There was no effect on the ABR of toluene alone. Noise alone produced a threshold shift. There was no interaction of noise and toluene on the ear. The present study suggests that chinchillas are markedly less susceptible to the ototoxic effect of toluene than mice and rats. A working hypothesis as to the species differences was that chinchilla liver was able to detoxify the toluene. Hepatic microsomes from chinchillas, rats and humans were tested for their ability to convert toluene to the more water-soluble compound - benzyl alcohol. Chinchilla livers were found to contain more of the P450 enzymes CYP2E1 and CYP2B than rats or humans. In addition, the data show that the P450 enzymes are more active in chinchillas than in rats and humans. In conclusion, the results suggest that rats and mice are a more appropriate model for human toluene ototoxicity. However, chinchillas may provide a valuable model for investigating how ototoxic agents can be detoxified to less damaging compounds.

Characterization of cytochrome P450 and glutathione S-transferase activity and expression in male and female ob/ob mice

OBJECTIVE: To characterize the effect(s) of gender, age (glycemic status) and obese state, on hepatic biotransformation activities, expression of cytochrome P450 (CYP450) mRNAs and glutathione transferase activity in the ob/ob mouse.DESIGN: Male and female, ob/ob or ob/+ mice were killed at 3–4 months or 7–8 months of age. Hepatic microsomes, cytosol and RNA were prepared from each animal.ANIMALS: Male and female ob/ob and ob/+ mice, 3–4 or 7–8 months of age.MEASUREMENTS: CYP450 form-specific activities of CYP1A1/1A2, CYP3A and CYP2B were estimated by determining the 0-dealkylation of alkoxyresorufin substrates (ethoxy-EROD, benzoxy-BROD and pentoxy-resorufin, PROD, respectively). CYP2E1-dependent, 4-nitrophenol hydroxylase (PNP-OH) and CYP3A-dependent erythromycin N-demethylase (ERY-DM) were also measured in hepatic microsomes. CYP1A2, CYP2E1 and CYP3A protein in microsomal fractions was determined by ELISA. Glutathione transferase activity (GST) was determined in hepatic cytosol and CYP1A2 and CYP2E1 mRNA was estimated by Northern blot analysis.RESULTS: Female mice, regardless of glycemic status, showed an obesity enhanced level of CYP2E1-dependent PNP-OH activity and CYP2EI protein as shown by ELISA. These increases were observed to be independent of the diabetic state, since 7–8 month-old mice had blood glucose levels identical to lean mice. The mRNA level of CYP2E1 in female mice also exhibited age-and obesity-influenced decreases in expression. No significant differences in CYP2E1 activity or expression were observed in male mice. CYP3A-dependent ERY-DM activity was significantly higher in young males, regardless of phenotype. CYP3A and CYP2B activities did not differ among any animals; however, CYP1A activity, while depressed in obese animals of both genders, was significantly different in old animals. Glutathione S-transferase activity was lower in obese male mice, whereas no difference was observed between lean and obese femalesCONCLUSION: This study supports earlier observations in man and rats that the obese state produces alterations in the expression of important oxidation and conjugation pathways. In addition, this report more thoroughly examines the role of gender and glycemic status on biotransformation activities in the ob/ob mouse as demonstrated by increased CYP2E1 protein and CYP2E1-dependent activity in obese females, decreased CYP1A2 protein and CYP1A2-dependent activity in obese animals, and obesity had no effect of glutathione transferase in female mice, in contrast with the previously reported obesity-dependent decrease of this activity in male mice.

Interlaboratory comparison of three multiplexed bead-based immunoassays for measuring serum antibodies to pneumococcal polysaccharides.

ABSTRACT Serotype-specific IgG, as quantified by a standardized WHO enzyme-linked immunosorbent assay (ELISA), is a serologic end point used to evaluate pneumococcal polysaccharide-based vaccine immunogenicity. Antibodies to each vaccine polysaccharide in licensed multivalent vaccines are quantified separately; this is laborious and consumes serum. We compared three bead-based immunoassays: a commercial assay (xMAP Pneumo14; Luminex) and two in-house assays (of the Health Protection Agency [HPA] and Centers for Disease Control and Prevention [CDC]), using the WHO-recommended standard reference and reference sera (n = 11) from vaccinated adults. Multiple comparisons of the IgG concentrations for seven conjugate vaccine serotypes were performed by sample (percent error), serotype (equivalency testing), and laboratory (concordance correlation coefficient [CCC]). When comparing concentrations by sample, bead-based immunoassays generally yielded higher antibody concentrations than the ELISA and had higher variability for serotypes 6B, 18C, and 23F. None of the three assays met the current WHO recommendation of 75% of sera falling within 40% of the assigned antibody concentrations for all seven serotypes. When compared by serotype, the CDC and HPA tests were equivalent for five of seven serotypes, whereas the Luminex assay was equivalent for four of seven serotypes. When overall mean IgG concentrations were compared by laboratory, a higher level of agreement (CCC close to 1) was found among bead-based immunoassays than between the assays and WHO assignments. When compared to WHO assignments, the HPA assay outperformed the other assays (r = 0.920; CCC = 0.894; coefficient of accuracy = 0.972). Additional testing with sera from immunogenicity studies should demonstrate the applicability of this methodology for vaccine evaluation.

Predictors of Airborne Exposures to Polycyclic Aromatic Compounds and Total Organic Matter among Hot-Mix Asphalt Paving Workers and Influence of Work Conditions and Practices

OBJECTIVES We evaluated personal airborne exposures to polycyclic aromatic compounds (PACs) and total organic matter (TOM) among hot-mix asphalt (HMA) paving workers. The primary objectives of this study were to identify predictors of airborne PAC exposures, identify PAC exposure sources, and characterize how work practices may affect personal airborne exposure to PACs. METHODS Four workers were recruited from each of three asphalt paving crews (12 workers) and were monitored for three consecutive days over 4 weeks for a total of 12 sampling days per worker (144 worker-days). Three sampling weeks were conducted while maintaining standard working conditions with regard to airborne exposures. The fourth week included the substitution of biodiesel for diesel oil used to clean tools and equipment. Linear mixed-effects models were used to evaluate predictors of airborne exposures including weather parameters (air temperature, wind speed, and relative humidity), worksite conditions (HMA application temperature, work rate, asphalt grade, and biodiesel use), and personal factors (minutes sampled, minutes of downtime, and smoking status). RESULTS Concentrations of the 33 individual PACs measured in personal air samples were generally below detection limits under all conditions with the exception of fluorene [geometric mean (GM) = 65 ng m(-3)], naphthalene (GM = 833 ng m(-3)), phenanthrene (GM = 385 ng m(-3)), and pyrene (GM = 57 ng m(-3)). The summary measures of TOM (GM = 864 μg m(-3)) and four- to six-ring PAC (GM = 0.13 μg m(-3)) were detected in the majority of air samples. Although task was not a predictor of airborne exposures, job site characteristics such as HMA application temperature were found to significantly (P ≤ 0.001) affect summary and individual PAC exposures. Based on the results of multivariate linear mixed-effects models, substituting biodiesel for diesel oil as a cleaning agent was associated with significant (P ≤ 0.01) reductions in TOM, four- to six-ring PACs, and naphthalene and pyrene concentrations that ranged from 31 to 56%. Using multivariate linear mixed-effects models under standard conditions, reducing the application temperature of HMA from 149°C (300°F) to 127°C (260°F) could be expected to reduce airborne exposures by 42-82%, varying by analyte. CONCLUSIONS Promising strategies for reducing airborne exposures to PACs among HMA paving workers include substituting biodiesel for diesel oil as a cleaning agent and decreasing the HMA application temperature.

Using Urinary Biomarkers of Polycyclic Aromatic Compound Exposure to Guide Exposure-Reduction Strategies Among Asphalt Paving Workers

INTRODUCTION Paving workers are exposed to polycyclic aromatic compounds (PACs) while working with hot-mix asphalt (HMA). Further characterization of the source and route of these exposures is necessary to guide exposure-reduction strategies. METHODS Personal air (n=144), hand-wash (n=144), and urine (n=480) samples were collected from 12 paving workers over 3 workdays during 4 workweeks. Urine samples were collected at preshift, postshift, and bedtime and analyzed for 10 hydroxylated PACs (1-OH-pyrene; 1-, 2-, 3-, 4-OH-phenanthrene; 1-, 2-OH-naphthalene; 2-, 3-, 9-OH-fluorene) by an immunochemical quantification of PACs (I-PACs). The air and hand-wash samples were analyzed for the parent compounds corresponding to the urinary analytes. Using a crossover study design, each of the 4 weeks represented a different exposure scenario: a baseline week (normal conditions), a dermal protection week (protective clothing), a powered air-purifying respirator (PAPR) week, and a biodiesel substitution week (100% biodiesel provided to replace the diesel oil normally used by workers to clean tools and equipment). The urinary analytes were analyzed using linear mixed-effects models. RESULTS Postshift and bedtime concentrations were significantly higher than preshift concentrations for most urinary biomarkers. Compared with baseline, urinary analytes were reduced during the dermal protection (29% for 1-OH-pyrene, 15% for I-PACs), the PAPR (24% for 1-OH-pyrene, 15% for I-PACs), and the biodiesel substitution (15% for 1-OH-pyrene) weeks. The effect of PACs in air was different by exposure scenario (biodiesel substitution>dermal protection>PAPR and baseline) and was still a significant predictor of most urinary analytes during the week of PAPR use, suggesting that PACs in air were dermally absorbed. The application temperature of HMA was positively associated with urinary measures, such that an increase from the lowest application temperature (121°C) to the highest (154°C) was associated with a 72% increase in ΣOH-fluorene and 1-OH-pyrene and an 82% increase in ΣOH-phenanthrene. Though PACs in hand-wash samples were not predictors of urinary analytes, the effects observed during the PAPR scenario and the week of increased dermal protection provide evidence of dermal absorption. CONCLUSIONS Our results provide evidence that PACs in air are dermally absorbed. Reducing the application temperature of asphalt mix appears to be a promising strategy for reducing PAC exposure among paving workers. Additional reductions may be achieved by requiring increased dermal coverage of workers and by substituting biodiesel for diesel oil as a cleaning agent.

Analytical Performance Criteria

The past five years have seen a move towards standardizing the documentation of measurement uncertainty through nearly worldwide adoption of the International Organization for Standardization Guide...