Isolation of a highly thermal stable lama single domain antibody specific for Staphylococcus aureus enterotoxin B.

BACKGROUND: Camelids and sharks possess a unique subclass of antibodies comprised of only heavy chains. The antigen binding fragments of these unique antibodies can be cloned and expressed as single domain antibodies (sdAbs). The ability of these small antigen-binding molecules to refold after heating to achieve their original structure, as well as their diminutive size, makes them attractive candidates for diagnostic assays. RESULTS: Here we describe the isolation of an sdAb against Staphyloccocus aureus enterotoxin B (SEB). The clone, A3, was found to have high affinity (Kd = 75 pM) and good specificity for SEB, showing no cross reactivity to related molecules such as Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin D (SED), and Shiga toxin. Most remarkably, this anti-SEB sdAb had an extremely high Tm of 85°C and an ability to refold after heating to 95°C. The sharp Tm determined by circular dichroism, was found to contrast with the gradual decrease observed in intrinsic fluorescence. We demonstrated the utility of this sdAb as a capture and detector molecule in Luminex based assays providing limits of detection (LODs) of at least 64 pg/mL. CONCLUSION: The anti-SEB sdAb A3 was found to have a high affinity and an extraordinarily high Tm and could still refold to recover activity after heat denaturation. This combination of heat resilience and strong, specific binding make this sdAb a good candidate for use in antibody-based toxin detection technologies.

Priming of familiar and unfamiliar visual objects over delays in young and older adults.

Although priming of familiar stimuli is usually age invariant, little is known about how aging affects priming of preexperimentally unfamiliar stimuli. Therefore, this study investigated the effects of aging and encoding-to-test delays (0 min, 20 min, 90 min, and 1 week) on priming of unfamiliar objects in block-based priming paradigms. During the encoding phase, participants viewed pictures of novel objects (Experiments 1 and 2) or novel and familiar objects (Experiment 3) and judged their left-right orientation. In the test block, priming was measured using the possible-impossible object-decision test (Experiment 1), symmetric-asymmetric object-decision test (Experiment 2), and real-nonreal object-decision test (Experiment 3). In Experiments 1 and 2, young adults showed priming for unfamiliar objects at all delays, whereas older adults whose baseline task performance was similar to that of young adults did not show any priming. Experiment 3 found no effects of age or delay on priming of familiar objects; however, priming of unfamiliar objects was only observed in the young participants. This suggests that when older adults cannot rely on preexisting memory representations, age-related deficits in priming can emerge.

Effects of dividing attention during encoding on perceptual priming of unfamiliar visual objects.

According to the distractor-selection hypothesis (Mulligan, 2003), dividing attention during encoding reduces perceptual priming when responses to non-critical (i.e., distractor) stimuli are selected frequently and simultaneously with critical stimulus encoding. Because direct support for this hypothesis comes exclusively from studies using familiar word stimuli, the present study tested whether the predictions of the distractor-selection hypothesis extend to perceptual priming of unfamiliar visual objects using the possible/impossible object decision test. Consistent with the distractor-selection hypothesis, Experiments 1 and 2 found no reduction in priming when the non-critical stimuli were presented infrequently and non-synchronously with the critical target stimuli, even though explicit recognition memory was reduced. In Experiment 3, non-critical stimuli were presented frequently and simultaneously during encoding of critical stimuli; however, no decrement in priming was detected, even when encoding time was reduced. These results suggest that priming in the possible/impossible object decision test is relatively immune to reductions in central attention and that not all aspects of the distractor-selection hypothesis generalise to priming of unfamiliar visual objects. Implications for theoretical models of object decision priming are discussed.

Evaluating models of object-decision priming: evidence from event-related potential repetition effects.

This study was designed to differentiate between structural description and bias accounts of performance in the possible/impossible object-decision test. Two event-related potential (ERP) studies examined how the visual system processes structurally possible and impossible objects. Specifically, the authors investigated the effects of object repetition on a series of early posterior components during structural (Experiment 1) and functional (Experiment 2) encoding and the relationship of these effects to behavioral measures of priming. In both experiments, the authors found repetition enhancement of the posterior N1 and N2 for possible objects only. In addition, the magnitude of the N1 repetition effect for possible objects was correlated with priming for possible objects. Although the behavioral results were more ambiguous, these ERP results fail to support bias models that hold that both possible and impossible objects are processed similarly in the visual system. Instead, they support the view that priming is supported by a structural description system that encodes the global 3-dimensional structure of an object.

Explicit and implicit memory for rotating objects.

Although both the object and the observer often move in natural environments, the effect of motion on visual object recognition ha not been well documented. The authors examined the effect of a reversal in the direction of rotation on both explicit and implicit memory for novel, 3-dimensional objects. Participants viewed a series of continuously rotating objects and later made either an old-new recognition judgment or a symmetric-asymmetric decision. For both tasks, memory for rotating objects was impaired when the direction of rotation was reversed at test. These results demonstrate that dynamic information can play a role in visual object recognition and suggest that object representations can encode spatiotemporal information.

Real-time PCR detection of salmonella in suspect foods from a gastroenteritis outbreak in kerr county, Texas.

In June 2001, an outbreak of acute gastroenteritis among 109 attendees of a church picnic in Kerr County, Texas, was reported. A 5'-nuclease PCR assay was used to screen for Salmonella in nine food items from the buffet line. Barbeque chicken B tested positive for Salmonella, and no amplification was detected in the remaining food items. These PCR findings were consistent with culture results and were confirmed by direct nucleotide sequencing. Salmonella enterica serotype Panama was cultured from both food and patient stool samples.

DNA vaccine expressing conserved influenza virus proteins protective against H5N1 challenge infection in mice.

Influenza vaccination practice, which is based on neutralizing antibodies, requires being able to predict which viral strains will be circulating. If an unexpected strain, as in the 1997 H5N1 Hong Kong outbreak, or even a pandemic emerges, appropriate vaccines may take too long to prepare. Therefore, strategies based on conserved influenza antigens should be explored. We studied DNA vaccination in mice with plasmids expressing conserved nucleoprotein (NP) and matrix (M) from an H1N1 virus. After vaccination, mice were challenged with A/H5N1 viruses of low, intermediate, and high lethality. A/NP+A/M DNA vaccination reduced replication of A/Hong Kong/486/97 (HK/486), a nonlethal H5N1 strain, and protected against lethal challenge with more virulent A/Hong Kong/156/97 (HK/156). After HK/156 exposure, mice survived rechallenge with A/Hong Kong/483/97 (HK/483), although the DNA vaccination alone protected poorly against this highly virulent strain. In the absence of antigenically matched hemagglutinin-based vaccines, DNA vaccination with conserved influenza genes may provide a useful first line of defense against a rapidly spreading pandemic virus.