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.

A newly recognized virus associated with a fatal case of hantavirus pulmonary syndrome in Louisiana.

Genetic analysis of virus detected in autopsy tissues of a fatal hantavirus pulmonary syndrome-like case in Louisiana revealed the presence of a previously unrecognized hantavirus. Nucleotide sequence analysis of PCR fragments of the complete S and M segments of the virus amplified from RNA extracted from the tissues showed the virus to be novel, differing from the closest related hantavirus, Sin Nombre virus, by approximately 30%. Both genome segments were unique, and there was no evidence of genetic reassortment with previously characterized hantaviruses. The primary rodent reservoir of Sin Nombre virus, the deer mouse Peromyscus maniculatus, is absent from Louisiana. Thus, the virus detected in Louisiana, referred to here as Bayou virus, must possess a different rodent reservoir.

[Laboratory diagnosis of pneumonia caused by Mycoplasma pneumoniae]. / Laboratornaia diagnostika pnevmonii, étiologicheski obuslovlennykh M. pneumoniae .

The authors demonstrated the possibility to use the technique of counter current immunoelectrophoresis, enzyme immunoassay and passive hemagglutination tests with antibody erythrocytic diagnosticum for the rapid laboratory diagnosis of Mycoplasma infection. The rapid diagnosis of Mycoplasma pneumonias turned to be possible due to the detection of antigen and antibodies to M. pneumoniae in the circulation with the help of counter current immunoelectrophoresis.

[HBs antigenemia in glomerulonephritis in children]. / HBs-antigenemiia pri glomerulonefritakh u detei.

Altogether 194 glomerulonephritis patients were examined by three methods: countercurrent immunoelectrophoresis, passive hemagglutination test, and enzyme immunoassay. Use of the most sensitive method, viz. enzyme immunoassay, has yielded the highest HBsAg detection rate: 29.1% in acute glomerulonephritis and 21.4% in chronic glomerulonephritis. This method may be recommended for the examination of glomerulonephritis patients whose sera contain HBsAg in low titers.

[Accelerated laboratory diagnosis of bacterial pneumonia]. / Uskorennaia laboratornaia diagnostika bakterial'nykh pnevmonii.

The effectiveness of countercurrent immunoelectrophoresis (CIE) used for the accelerated differential diagnosis of pneumococcal, staphylococcal, mycoplasmal and Legionella infections in cases of pneumonia has been shown. The presence of correlation between the results obtained in the bacterial study of sputa and bronchial washings and in CIE has been revealed, which gives grounds for recommending CIE for the accelerated diagnosis of pneumococcal pneumonia on the basis of the analysis of sputa, bronchial washings and blood sera obtained from patients. In Legionella infection the passive hemagglutination test with antigenic diagnostica has proved to be more effective for accelerated diagnosis than CIE.

[Activity of the glycoprotein antigenic fractions of Mycoplasma pneumoniae in immunological reactions]. / Izuchenie aktivnosti glikoproteidnykh antigennykh fraktsii Mycoplasma pneumoniae v immunologicheskikh reaktsiiakh.

Glycoprotein fractions exhibiting pronounced antigenic properties in immunological in vitro reactions have been isolated from sonicated M. pneumoniae cells by extraction with glacial acetic acid and ethanol. Glycoprotein fraction 3 of M. pneumoniae antigen shows the highest blood-sensitive and precipitating activity and may be recommended as a diagnosticum for serological tests.

[Etiological structure of pneumonias in children and adults]. / Etiologicheskaia struktura pnevmonii u detei i vzroslykh.

The bacteriological study of sputa, nasopharyngeal smears and bronchial washings taken from pneumonia patients has shown that the leading etiological agent was Streptococcus pneumoniae isolated in the diagnostic titre (10(7) bacteria per ml) in 78.1% of the cases. Staphylococcus aureus, Haemophilus influenzae, enterobacteria and yeast-like fungi have been found to play an insignificant role in the etiology of acute pneumonia (2.5 +/- +/- 0.9%). The results of the serological diagnosis by means of the complement fixation test have revealed that, alongside S. pneumoniae, the following infective agents are of etiological importance in cases of acute pneumonia: respiratory viruses (more than 50%), Mycoplasma pneumonia (10%), Chlamydia psittaci (6.4%) and Legionella pneumophila (3.8%). The study has first revealed that, under the conditions of Alma-Ata, serotypes 19, 23, 8 and 4 prevail among circulating pneumococci. This study has also shown that the use of M. pneumoniae antibody erythrocyte diagnosticum enhances the detection rate of mycoplasma infections in pneumonia patients.

[Circulating immune complexes in the diagnosis of Mycoplasma pneumoniae infection]. / Issledovanie tsirkuliruiushchikh immunnykh kompleksov pri diagnostike infektsii Mycoplasma pneumoniae.

The possibility of detecting M. pneumoniae antigen and antibodies to it, incorporated into immune complexes, in the sera of patients with acute pneumonia by means of erythrocyte diagnosticums was studied, and the immunological characterization of these complexes was made. In patients with mycoplasmal pneumonia M. pneumoniae antigen and specific antibodies, both free and incorporated into immune complexes, were found to circulate in the blood. In children, antigenemia was detected twice as frequently as in adults. Dissociated M. pneumoniae antigens had different molecular weight, their location on the gel chromatogram of the serum being in fractions 7S and 19S. The dissociation of immune complexes permits the detection of M. pneumoniae antigen and antibodies to it in a bound state by means of the passive hemagglutination test, thus increasing the frequency of positive results in the diagnosis of M. pneumoniae infection.

[Serum immunoglobulin in Mycoplasma pneumoniae infection]. / Izuchenie syvorotochnykh immunoglobulinov pri infektsii Mycoplasma pneumoniae.

The dynamics and nature of serum antibodies in experimental and natural M. pneumoniae infection have been studied. The synthesis of specific IgM, IgA and IgG has been found to occur in the course of infection. During repeated M. pneumoniae infection in guinea pigs, as well as in cases of acute and chronic mycoplasmal pneumonia in humans (at the acute period of the disease), the prevalence of the synthesis of specific IgM is observed. At the acute period of the disease a rise in the quantitative levels of IgM and IgG occurs in patients. High titers of specific IgM (1:32 and greater) determined at the acute period of the disease can serve as the diagnostic criterion of the mycoplasmal etiology of pneumonia.

Analysis of anabolic steroids in body fluids by capillary gas chromatography with a two-channel detection system and a computer.

A method is described for analysis of multi-component mixtures of steroid metabolites in biological fluids by means of capillary gas chromatography with glass and fused-silica columns and simultaneous detection of methoxylamine-trimethylsilyl derivatives with universal flame-ionization and selective nitrogen alkali flameionization detectors. A data system was applied to the on-line treatment of the results. Computer programs were designed for precise calculation of Kováts retention indices from the known values for selected natural urinary steroids. The programs allow the selection of nitrogen-containing components, normalized chromatogram plotting for both detection channels and qualitative and quantitative analysis. Results are presented on the detection of metabolites of methandrostenolone, 17 alpha-methyltestosterone, 19-nortestosterone and fluoxymesterone.