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.

Co-design in synthetic biology: a system-level analysis of the development of an environmental sensing device.

The concept of co-design is common in engineering, where it is necessary, for example, to determine the optimal partitioning between hardware and software of the implementation of a system features. Here we propose to adapt co-design methodologies for synthetic biology. As a test case, we have designed an environmental sensing device that detects the presence of three chemicals, and returns an output only if at least two of the three chemicals are present. We show that the logical operations can be implemented in three different design domains: (1) the transcriptional domain using synthetically designed hybrid promoters, (2) the protein domain using bi-molecular fluorescence complementation, and (3) the fluorescence domain using spectral unmixing and relying on electronic processing. We discuss how these heterogeneous design strategies could be formalized to develop co-design algorithms capable of identifying optimal designs meeting user specifications.

Asbestos burden in cases of mesothelioma from individuals from various regions of the United States.

Mesothelioma is a rare tumor that is considered an asbestos marker disease. It occurs in individuals following a longer latency period from first exposure than other asbestos-related diseases. The tumor also occurs in individuals with a wide range of exposures, including individuals with lower level or secondary exposures. In the present study lung tissue from 54 individuals with a pathological diagnosis of mesothelioma was evaluated for ferruginous body and uncoated asbestos fiber content. The data were compared with an earlier study of mesothelioma cases from the northwestern United States. Tissue was prepared via a digestion procedure, with the collected digestate reviewed by light microscopy for quantification of asbestos bodies and analytical transmission electron microscopy for determination of uncoated fiber burden. Twenty-seven cases in the present study had over 1000 ferruginous bodies per gram of dry tissue. The data suggest that amosite provides a more likely stimulus for ferruginous coating than the other forms of asbestos. All individuals were found to have asbestos fibers in their lung tissue. Amosite was the most commonly found fiber, with anthophyllite being the second most commonly found type of asbestos. The finding of tremolite in the tissue most often was associated with the finding of anthophyllite. A limited number of asbestos fibers of each type would have been seen in the light microscope, with the least detected being chrysotile. The majority of all fiber types were found as short fibers (< 8 mum), although some longer fibers were represented in each type of asbestos. The majority of the individuals were found to have mixed types of asbestos in their lungs.