Raymond E. Biagini

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.

Method for Simultaneous Measurement of Antibodies to 23 Pneumococcal Capsular Polysaccharides

ABSTRACT We describe a fluorescent covalent microsphere immunoassay (FCMIA) method for the simultaneous (multiplexed) measurement of immunoglobulin G (IgG) antibodies to 23 pneumococcal capsular polysaccharide (PnPS) serotypes present in the pneumococcal polysaccharide vaccine (PPV23) licensed by the Food and Drug Administration, i.e., PnPSs 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. In addition, the assay incorporates an internal control that allows for contemporaneous evaluation of the effectiveness of pneumococcal cell wall polysaccharide (C-PS) preadsorption and a second control of PnPS 25 (which is not present in any polysaccharide or conjugate vaccine), which can be used to evaluate interassay reproducibility (useful for pre- versus postvaccination studies). The FCMIA was standardized with U.S. reference antipneumococcal serotype standard serum 89S-2. Preadsorption of 89S-2 with each PnPS and C-PS yielded homologous inhibition for serotypes 1, 6B, 9N, 9V, 11A, 12F,14, 15B, 18C, 19A, 19F, 20, 22F, 25, and 33F; heterologous inhibition for serotypes 9V, 10A, 11A, 12F, 15B, 17F, 20, and 23F; and neither homologous nor heterologous inhibition for serotypes 2, 3, 4, and 5. The minimum detectable concentrations for the 24 multiplexed (PnPS and C-PS) FCMIAs ranged from 20 pg/ml for PnPS 3 to 600 pg/ml for PnPS 14. The PnPS FCMIA method has numerous benefits over enzyme-linked immunosorbent assays commonly used to measure anti-PnPS-specific IgG levels, including increased speed, smaller sample volumes, equivalent or better sensitivity, and increased dynamic range.

The diversity of reaginic immune responses to platinum and palladium metallic salts

As part of a National Institute for Occupational Safety and Health health hazards evaluation, workers employed in a precious metal refinery exposed to platinum (Pt), palladium (Pd), and other group VIII metallic salts were evaluated for direct skin test sensitivity to Pt. Current (107) and former (30) workers who quit or were discharged because of Pt-related health problems were prick tested with ammonium hexachloroplatinate ([NH4]2 PtCl6). Of the 107 currently exposed workers, 15 (14%) exhibited positive skin tests, as indexed by immediate reactivity at a dose of 10(-3) gm/ml or less. Eight (27%) of the 30 former workers no longer exposed to Pt also demonstrated positive Pt skin tests. Sera obtained from the workers were assessed for transferable antibodies to Pt and Pd salts by monkey passive cutaneous anaphylaxis. In addition, Pt-specific antibodies were evaluated by RAST. Results of these studies suggested that short- and long-term passive cutaneous anaphylaxis immune responses occur after exposure to both Pt and Pd compounds. Results of RAST analysis for Pt-specific antibodies indicated that significantly higher (p less than 0.001) levels were present in the sera of skin test-positive workers as compared to control sera from Pt-exposed, skin test-negative workers or nonexposed control subjects. Evidence was also obtained that Pt or Pt-protein adducts present in the sera of exposed workers may compete for IgE-binding sites in the RAST assay. The specificity of the Pt-specific RAST system was proved by inhibition experiments.

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.

Antibodies to morphine in workers exposed to opiates at a narcotics manufacturing facility and evidence for similar antibodies in heroin abusers

According to the International Narcotics Control Board, over 45,000 kg of morphine and 54,000 kg of codeine were ethically manufactured in 1986 at three facilities in the United States. Little information exists about possible adverse health effects associated with workplace exposure to opiate compounds in this industry. Because there are no specific federal standards for workplace exposure to narcotic dusts, exposure-control defaults to the nuisance dust standard (10 mg/m3, as an 8 hr time-weighted average). Narcotics manufacturing workers were evaluated for anti-morphine IgG before and 10 mo. after the implementation of an improved respiratory protection program (RPP). Significantly elevated IgG levels were measured before the improved RPP (P less than 0.005). After the improved RPP, a significant reduction was observed (P less than 0.001), suggesting that specific antibody levels could be used as biomarkers of exposure. Inhibition studies showed that the antibodies were specifically directed against morphine with some cross reactivity with morphine derivatives. Preliminary results are also shown which indicate that similar anti-morphine antibodies are present in the sera of intravenous heroin abusers. Elevated levels (P less than 0.05) of anti-morphine antibodies were detected in sera from heroin abusers, providing evidence that similar antibodies may be produced from non-occupational exposure to opiates. These finding have potentially far-reaching implications for addiction research and drug testing.

Immunologic findings among lead-exposed workers

A comprehensive panel of immune parameters was evaluated among 145 lead-exposed workers with a median blood lead level (BLL) of 39 micrograms/dL (range: 15-55 micrograms/dL) and 84 unexposed workers. After adjusting for covariates, we found no major differences in the percentage of CD3+ cells, CD4+ T cells, CD8+ T cells, B cells, or NK cells between lead-exposed and unexposed workers, although the association between lead exposure and the number of CD4+ T cells was modified by age. We also found no differences between exposed and unexposed workers in serum immunoglobulin levels, salivary IgA, C3 complement levels, or lymphoproliferative responses. However, among exposed workers, the percentage and number of B cells were positively associated with current BLL, serum IgG was negatively associated with cumulative lead exposure, and the percentage and number of CD4+/CD45RA+ cells were positively associated with cumulative lead exposure. We found no evidence of a marked immunotoxic effect of lead at the exposure levels studied, although some subtle differences in immunologic parameters were noted.

Inhibition of rat heart mitochondrial electron transport in vitro: implications for the cardiotoxic action of allylamine or its primary metabolite, acrolein

Allylamine (3-aminopropene) is a specific cardiac toxicant that causes aortic, valvular and myocardial lesions in many species. Myocardial necrosis can be observed 24 h after a single dose. Acute toxicity is believed to involve metabolism of allylamine to highly reactive acrolein (2-propenal). Allylamine has been shown to bind to mitochondria from aorta and heart, suggesting that the subcellular site of injury is at or near the mitochondrion. The present investigation compared the effect of allylamine and its primary metabolite, acrolein, on electron transport and oxidative phosphorylation in mitochondria isolated from rat heart (RHM). Both compounds weakly inhibited mitochondrial electron transport with either the combination of glutamate, malate, and malonate (GMM, NADH-linked) or succinate as substrate. Comparisons of the slopes of concentration-effect regression (range of concentrations tested, 0.20-2.0 mM) lines showed acrolein to have significantly greater inhibitory effects than allylamine (range of concentrations tested, 0.22-6.4 mM) on GMM oxidation, while no significant difference in the abilities of the compounds to inhibit succinate oxidation were observed, indicating site preferences for inhibitory action. The addition of an uncoupling agent could not reverse inhibition with either substrate system. These results indicate that both the parent compound and its proposed metabolite primarily inhibit electron transport with little direct effect on the coupling mechanism. The State III EC50 (effective concentrations for 50% inhibition of control mitochondrial enzyme activities) for allylamine (2.29 mM with succinate as substrate and 1.22 mM with GMM) and acrolein (0.80 mM with succinate as substrate and 0.39 mM with GMM) are probably too great to invoke the direct action of either the parent compound or its oxidized metabolite on mitochondrial electron transport as a primary mechanism in the cardiotoxic action of allylamine.

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.

Latex specific IgE: performance characteristics of the IMMULITE 2000 3gAllergy assay compared with skin testing

BACKGROUND In the absence of a US Food and Drug Administration (FDA)-cleared latex skin testing reagent, in vitro tests remain important for the diagnosis of latex allergy. OBJECTIVE To evaluate the performance characteristics of IMMULITE 2000 3gAllergy (Immulite), a third-generation, FDA-cleared, continuous random-access immunoanalyzer, for the quantification of latex specific IgE. METHODS Stored serum samples (N = 201) from patients classified as having positive or negative latex puncture skin test results were measured for latex specific IgE levels using Immulite, and these data were compared with historical results from 3 second-generation, FDA-cleared IgE antilatex assays (AlaSTAT [Ala], AutoCAP [CAP], and HY*TEC enzyme immunoassay [HT]). RESULTS The diagnostic performances of the CAP, Ala, and Immulite assays (> or = 0.35 kU/L cutoff value) were equivalent in sensitivity and specificity (P > .05). The HT assay (> or = 0.05 kU/L cutoff value) was more sensitive and less specific (P < .05). Immulite (> or = 0.10 kU/L cutoff value) had greater sensitivity than Ala and CAP and greater specificity than HT (P < .05 for both). Diagnostic efficiency was greater for Immulite than for CAP, Ala, and HT (P < .05). CONCLUSIONS The Immulite system is superior in diagnostic performance, especially at the 0.10 kU/L or greater cutoff level, for the diagnosis of latex allergy compared with older, second-generation assays. Immulite still misclassifies 15.5% of puncture skin test-positive individuals as negative for latex specific IgE. Compared with second-generation assays, Immulite represents a technological advance, with enhanced speed and less operator intervention.

Exposure to Protein Aeroallergens in Egg Processing Facilities

Proteinaceous materials in the air can be highly allergenic and result in a range of immunologically mediated respiratory effects, including asthma. We report on the largest evaluation of exposure to date of airborne egg protein concentrations in an egg breaking and processing plant that had cases of occupational asthma. Personal air samples for egg protein were analyzed in duplicate on each PTFE filter using two analytical methods: (1) a commercial assay for non-specific total protein, and (2) indirect competitive inhibition assay using an ELISA method to quantify specific egg protein components. The highest concentrations were found in the egg washing room (mean exposure 644 microg/m3) and breaking room (255 microg/m3), which were also the areas where the risk of being sensitized was the greatest. There was excellent quantitative agreement between the airborne concentrations of total protein and sum of the specific protein antigens (ovalbumin, ovomucoid, and lysozyme). The correlation coefficient of the log-transformed data from the two methods was 0.88 (p < 0.0001). Size-selective sampling also indicated that most of the aerosol was capable of reaching the small airways. The methods described can be utilized to evaluate employee exposure to egg proteins. Exposure documentation, coupled with recommended exposure reduction strategies, could facilitate prevention of future employee sensitization and allergic respiratory responses by identifying high-exposure jobs and evaluating control measures.