Evaluation of an immunochromatographic test for rapid and reliable serodiagnosis of human tularemia and detection of Francisella tularensis-specific antibodies in sera from different mammalian species.

Tularemia is a highly contagious infectious zoonosis caused by the bacterial agent Francisella tularensis. Serology is still considered to be a cornerstone in tularemia diagnosis due to the low sensitivity of bacterial culture and the lack of standardization in PCR methodology for the direct identification of the pathogen. We developed a novel immunochromatographic test (ICT) to efficiently detect F. tularensis-specific antibodies in sera from humans and other mammalian species (nonhuman primate, pig, and rabbit). This new tool requires none or minimal laboratory equipment, and the results are obtained within 15 min. When compared to the method of microagglutination, which was shown to be more specific than the enzyme-linked immunosorbent assay, the ICT had a sensitivity of 98.3% (58 positive sera were tested) and a specificity of 96.5% (58 negative sera were tested) on human sera. On animal sera, the overall sensitivity was 100% (22 positive sera were tested) and specificity was also 100% (70 negative sera were tested). This rapid test preferentially detects IgG antibodies that may occur early in the course of human tularemia, but further evaluation with human sera is important to prove that the ICT can be a valuable field test to support a presumptive diagnosis of tularemia. The ICT can also be a useful tool to monitor successful vaccination with subunit vaccines or live vaccine strains containing lipopolysaccharide (e.g., LVS) and to detect seropositive individuals or animals in outbreak situations or in the context of epidemiologic surveillance programs in areas of endemicity as recently recommended by the World Health Organization.

Comparison of an electrochemiluminescence assay in plate format over a colorimetric ELISA, for the detection of ricin B chain (RCA-B).

An electrochemiluminescence (ECL) assay for the detection of the B chain of ricin (RCA-B) in a 96-well plate format was developed in parallel with a colorimetric ELISA utilizing the same pair of antibodies. Sensitivity results were interpreted with the ANOVA and Tukey statistical tests, allowing a direct comparison between the two technologies, that can probably be extended to other protein antigens such as toxins. Reproducibility, repeatability and rapidity of the two techniques were also compared. The ELISA assay utilized an alkaline phosphatase conjugate for signal generation. After optimization, its limit of detection was 400 pg of RCA-B per ml buffer, with an intra-day standard deviation (SD) of 2.2% of the mean and an inter-day SD of 5.1%. The ECL assay utilized ruthenylated antibodies for detection. The ECL measurement was carried out using a Sector PR 400 plate reader. After optimization, its limit of detection was 50 pg of RCA-B per ml buffer, with an intra-day SD of 4.1% of the mean and an inter-day SD of 4.3%. Starting from a pre-coated plate, the ELISA assay was completed in 7 h and the ECL assay took 2.5 h. While reproducibility and repeatability of the two assays were equivalent, this ECL assay in plate format had an 8-fold better sensitivity for RCA-B detection than the colorimetric ELISA in buffer and in various matrices. The ECL assay was also three times faster, and retained the robustness and convenience of the 96-well plate format.

Comparison of electrochemiluminescence assay and ELISA for the detection of Clostridium botulinum type B neurotoxin.

We compared the ELISA and electrochemiluminescence (ECL) immunoassay technologies for the detection of botulinum type B neurotoxin (BotNT B), which requires highly sensitive techniques due to its potent biological activity. BotNT B complexes are the naturally secreted form of the toxin, approximately a third of which consists of the neurotoxin itself; they were aliquoted and frozen for this study. Results of both techniques were interpreted with the same standard statistical tests (ANOVA and Tukey). We first compared two commercial assays for BotNT B: the detection limit of the colorimetric ELISA was 1.56 ng/ml BotNT B complexes versus 0.39-0.78 ng/ml in the ECL test. We then used the same monoclonal antibody and the same polyclonal antibody, respectively purified by protein A and protein G chromatography, to optimize an in-house ELISA test and an in-house ECL test, making it possible to directly compare the two technologies without interference due to the properties of the antibodies used in the two tests. The colorimetric in-house ELISA had a detection threshold of 3.12 ng/ml versus the in-house ECL test whose detection threshold was 0.78-1.56 ng/ml. Thus, in both cases, the ECL assay was two to four times more sensitive than the colorimetric ELISA. The ECL assay was also more rapid (2.5 h for the in-house ECL versus 5 h for in-house ELISA with precoated wells). Overall, these elements can be used to compare the qualities of the two technologies, at least for the detection of protein antigens such as toxins.

Nonhuman primates are relevant models for research in hematology, immunology and virology.

Nonhuman primates have been used for biomedical research for several decades. They have proved to be models that are relevant to humans because of the high level of gene homology which underlies physiological and biochemical similarities. The similarity of monkeys to humans has been used to investigate pathophysiological mechanisms in hematology, immunology and virology. New therapeutic procedures can be assessed in primates by using materials, in particular pharmacological reagents, and methods designed for humans. The relevance of these models also relies on the use of species-specific pathogens and the availability of recombinant, homologous cytokines. The introduction of more and more sophisticated cell and gene therapy protocols in hematopoietic cell transplantation and immunotherapy requires the development of preclinical trials similar to clinical settings. For several decades now, baboons and cynomolgus/rhesus monkeys have been the most useful primate models in experimental hematology, and this has contributed to numerous therapeutic advances. Primate models of AIDS have been developed to study the pathogenesis, transmission and immune responses to infection, and to test vaccines and drugs. Primate research should be restricted in quantity, and mainly designed with the aim of removing uncertainty as to the safety and clinical benefit to the patient, of new biomedical protocols.

Simulation of cardiovascular response to lower body negative pressure from 0 to -40 mmHg.

This paper presents a mathematical model for simulation of the human cardiovascular response to lower body negative pressure (LBNP) up to -40 mmHg both under normal conditions and when arterial baroreflex sensitivity or leg blood capacity (LBC) is altered. Development of the model assumes that the LBNP response could be explained solely on the bases of 1) blood volume redistribution, 2) left ventricular end-diastolic filling, 3) interaction between left ventricle and peripheral circulation, and 4) modulations of peripheral resistances and heart rate by arterial and cardiopulmonary baroreflexes. The model reproduced well experimental data obtained both under normal conditions and during complete autonomic blockade; thus it is validated for simulation of the cardiovascular response from 0 to -40 mmHg LBNP. We tested the ability of the model to simulate the changes in LBNP response due to a reduction in LBC. To assess these changes experimentally, six healthy men were subjected to LBNP of -15, -30, and -38 mmHg with and without wearing elastic compression stockings. Stockings significantly reduced LBC (from 3.9 +/- 0.3 to 1.8 +/- 0.4 ml/100 ml tissue at -38 mmHg LBNP; P < 0.01) and attenuated the change in heart rate (from 23 +/- 4 to 8 +/- 3% at -38 mmHg LBNP; P < 0.05). The model accurately reproduced this result. The model is useful for assessing the influence of LBC or other parameters such as arterial baroreflex sensitivity in diminishing the orthostatic tolerance of humans after spaceflight, bed rest, or endurance training.

A recombinant Fab neutralizes dengue virus in vitro.

A recombinant Fab that recognizes a neutralizing epitope located in the (296-400) region of protein E of dengue virus was obtained from cloned hybridoma cells secreting the mouse monoclonal antibody (mAb) 4E11. The Fd and light chain antibody genes were amplified by polymerase chain reaction, cloned into the phagemid vector pMad, expressed in bacteria to produce Fab fragments and sequenced. The mAb 4E11, in particular its light chain complementary-determining regions, shared homologies with two other anti-viral mAbs. The affinity of the parental mAb and the cloned Fab to the MalE-E(296-400) fusion protein were shown to be of the same magnitude, i.e. nanomolar. Fab 4E11 neutralization capacity was found between 8 and 4-times or less lower than that of mAb 4E11, depending on serotypes, thus the Fab could have a smaller antiviral activity than the mAb in vitro.

Increase of LBNP tolerance using elastic compression stockings.

BACKGROUND: Astronauts returning from spaceflight are affected by reduced orthostatic tolerance resulting from exposure to weightlessness. There are some countermeasures currently in use to improve cardiovascular performance of returning astronauts, while there are others that are being tested in flight and in ground-based investigations. This paper presents a study on the use of elastic compression stockings to reduce leg blood capacity (LBC) which is believed to be one of the determinants of orthostatic tolerance. METHODS: The data are from 6 healthy men with a mean age of 36 +/- 5 (SE) yr. Assessment of the effectiveness of stockings in improving orthostatic tolerance is based on a presyncopal-limited lower body negative pressure (LBNP) test, consisting of successive 3 min exposures to negative pressures of -20 hPa (-15 mmHg), -40 hPa (-30 mmHg), and decrements in steps of 10 hPa (7.5 mmHg) from then on until termination of the test. RESULTS: Results show an increase in the maximal level of LBNP tolerated (88 hPa or 66 mmHg for control vs. 108 hPa or 81 mmHg for stockings; p = 0.018) as well as in the cumulative stress index (CSI) (1122 hPa-min or 842 mmHg-min for control vs. 1734 hPa-min or 1300 mmHg-min for stockings; p = 0.029). CONCLUSIONS: The improvement of LBNP tolerance with elastic compression stockings coupled with their ease of use support the need for further experimental studies for evaluating their potential as a countermeasure for astronauts after return from spaceflight.

Comparison of hand-held test kits, immunofluorescence microscopy, enzyme-linked immunosorbent assay, and flow cytometric analysis for rapid presumptive identification of Yersinia pestis.

An in-house immunochromatographic test, Plague BioThreat Alert test strips, ABICAP columns, enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence microscopy were compared for the detection of the fraction 1 capsular antigen of Yersinia pestis, using spiked buffer and clinical specimens. Hand-held test kits proved to be excellent benchtop tools.