Validation and long term performance characteristics of a quantitative enzyme linked immunosorbent assay (ELISA) for human anti-PA IgG.

Accurate, reliable and standardized quantification of anti-protective antigen (PA) IgG antibody levels is essential for comparative analyses of anti-toxin immune responses in anthrax cases, recipients of PA-based anthrax vaccines and for evaluation of anti-PA based immunotherapies. We have previously reported the early performance characteristics and application of a quantitative anti-PA IgG enzyme linked immunosorbent assay. The principal application of this assay was in a Phase 4 human clinical trial of anthrax vaccine adsorbed (AVA, BioThrax), the central component of the CDC Anthrax Vaccine Research Program (AVRP) and in humans following bioterrorism associated Bacillus anthracis infection (Quinn et al., 2002; Quinn et al., 2004; Marano et al., 2008). The objective of the AVRP was to determine the feasibility of reducing the number of priming series and booster doses of the licensed Anthrax Vaccine Adsorbed (AVA) (BioThrax®; Emergent BioSolutions, Lansing, MI) and changing the route of administration from subcutaneous (SC) to intramuscular (IM) (Marano et al., 2008). In this paper we report the validation and long term performance characteristics of the assay during its six year application in the AVRP (2002-2008). The critical features are 1) extensive validation of the assay using two standard reference sera; 2) long term stability and 3) consistency of the data for quantitative analysis of human long term anti-PA IgG responses. The reportable value (RV) of the assay was expressed as anti-PA IgG concentration (µg/ml). Accuracy of the assay was high with a percent error (%ER) range of 1.6-11.4%. Overall intra-operator and intermediate precision were high with Coefficients of Variation (%CVs) of 2.5-15.4% and 6.3-13.2%, respectively. The assay demonstrated excellent dilutional linearity for human sera using log(10) transformed data with the slope=0.95 to 0.99, intercept=0.02 to 0.06 and r(2)=0.980-0.987. The assay was robust, tolerating changes in serum incubation temperatures from 35 to 39°C, serum incubation times from 55 to 65min and changes in key reagents. The long-term assay stability over 6years using consecutive reference sera AVR414 and AVR801 demonstrated sustained high accuracy and precision for the assay, confirming its suitability for long term studies of PA protein-based anthrax vaccines.

Analysis of anti-protective antigen IgG subclass distribution in recipients of anthrax vaccine adsorbed (AVA) and patients with cutaneous and inhalation anthrax.

The anti-PA IgG1, IgG2, IgG3, and IgG4 subclass responses to clinical anthrax and to different numbers of anthrax vaccine adsorbed (AVA, BioThrax) injections were determined in a cross-sectional study of sera from 63 vaccinees and 13 clinical anthrax patients. The data show that both vaccination with three AVA injections and clinical anthrax elicit anti-PA IgG1, IgG2, and IgG3 subclass responses. An anti-PA IgG4 response was detected in AVA recipients after the fourth injection. The anthrax lethal toxin (LTx) neutralization efficacy of sera from recipients who received 4 to > or =10 AVA injections did not vary significantly in relation to changes in distribution of anti-PA IgG1 and IgG4 subclasses.

Mass value assignment of total and subclass immunoglobulin G in a human standard anthrax reference serum.

An anti-Anthrax Vaccine Adsorbed (anti-AVA) standard human reference serum pool, AVR414, has been prepared, and the total and protective antigen (PA)-specific immunoglobulin G (IgG) were quantified. AVR414 was prepared by plasmapheresis of healthy adults who had received a minimum of four subcutaneous injections of AVA. Mass values (in milligrams per milliliter) for total IgG and IgG subclasses 1 to 4 were determined by radial immunodiffusion. Anti-PA-specific IgG assignment (in micrograms per milliliter) was done by consensus of two complementary approaches: homologous enzyme-linked immunosorbent assay (ELISA) with affinity-purified anti-PA IgG as a calibrator and summation of mean PA-specific IgG subclass concentrations determined by IgG subclass-specific ELISA using the United States National Reference Preparation for Human Serum Proteins as a standard. The total IgG concentration assigned to AVR414 reference serum was 8.33 mg/ml. IgG subclass concentrations were the following: for IgG1, 4.48 mg/ml; for IgG2, 3.35 mg/ml; for IgG3, 0.37 mg/ml; and for IgG4, 0.30 mg/ml. The assigned mass value for total anti-PA-specific IgG was 141.2 microg/ml. Anti-PA-specific IgG subclass concentrations were the following: for IgG1, 79.6 microg/ml; for IgG2, 35.3 microg/ml; for IgG3, 3.2 microg/ml; and for IgG4, 25.3 microg/ml. Human reference serum pool AVR414 will have direct application in the standardization of anthrax serological assays, in reagent qualification, and as a standard for quantification of PA-specific IgG in humans who have been vaccinated with or otherwise exposed to Bacillus anthracis PA.

Random amplified polymorphic DNA analysis of Ustilago violacea.

Isolates of the two mating type strains of the basidiomycete phytopathogen Ustilago violacea (Pers.) Roussel [a.k.a Microbotryum violaceum (Pers.:Pers.) Deml and Oberw] are restricted in their host range to one or a few species of Caryophyllaceae (Pinks). Molecular genetics maps in this species are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs) among the progeny of a sexual cross and more recently through electrophoretic karyotypes and chromosomal polymorphism using CHEF gel analysis. However, currently, polymorphisms in genomic fingerprints generated by arbitrarily primed polymerase chain reaction (PCR) can distinguish between strains of almost any organism, which is useful in genetic mapping. The objective of this project was to use PCR technology, 40 Operon 10-mer primers, and 5 simple sequence repeat (SSR) primers, designed on microsatellite sequences to determine their efficiency in detecting intraspecific differences between the genomic DNA of the two mating type strains of U. violacea (a1 and a2). Polymorphism in the banding patterns of the DNA samples was detected after PCR and electrophoresis in 1.4% agarose gels. This polymorphic intraspecific variation will be utilized to detect cryptic and trans-active transposable elements in U. violacea.