Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens

The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv) in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.     

The use of lyophilized Schistosoma mansoni eggs as antigenic particles in a radioimmunoassay

Lyophilized eggs of Schistosoma mansoni, when incubated briefly with serum from infected mice, bind antibodies, as made evident by subsequent binding of fluorescein labelled anti-IgG or 125I-labelled Protein A. On the basis of these findings, a radioimmunoassay was devised which employs whole lyophilized eggs (500 or 250 eggs/serum sample) as antigenic particles and 125I-labelled Protein A as a probe for antibody binding. Only 10 microliters of serum are required to obtain 90% of the maximal binding. Kinetic studies indicated that 70% of the maximal seropositivity develops in mice between five and six weeks after a light infection, reaches a maximum at eight weeks and fluctuates around a high plateau thereafter. Pre-incubation of the test serum with soluble egg antigen (SEA) considerably inhibits antibody binding to the eggs, suggesting that SEA-like antigens participate in the reaction.     

Quantifying the effect of brush layering on slope stability

Soil bioengineering techniques that use vegetation as a structural element gained popularity in the field of natural and man-made slope stabilisation due to their ability to combine safety and environmental conservation elements. In spite of such popularity, little research has been done to quantify their effect on slope stability. This work presents a simple scheme for the evaluation of the Factor of Safety for slopes reinforced by brush layering, which is one of the most common techniques adopted in slope stabilisation works. The proposed model is based on the limit equilibrium principle and accounts for geotechnical soil properties (cohesion, friction angle, unit weight of soil), soil saturation, slope steepness, and brush layer design parameters (number of stems per meter, length and diameter of stems, distance between brush layers). The model provides the value of the Factor of Safety for a given slope and soil depth. Laboratory pullout tests were carried out in order to estimate relevant parameters of cuttings of purple willow (Salix purpurea L.) and to perform a slope stability analysis via the model.     

The use of enhanced polymer one-step staining reagents for immunoenzyme double-labelling

Summary The newly developed peroxidase-labelled Enhanced Polymer One-Step (EPOS) reagents were applied, together with an unlabelled primary mouse antibody, in a multistep double-labelling protocol. Enzyme label reporter combinations consisted of either peroxidase and alkaline phosphatase in red and blue, respectively, or β-galactosidase and alkaline phosphatase in turquoise and red, respectively. The latter enzyme combination was introduced using a rabbit antiperoxidase antibody and an enzyme-labelled anti-rabbit immunoglobulin antibody. The multistep procedure was tested using five different antibody combinations on cryostat and Carnoy- or formalin-fixed, paraffin-embedded sections. In each instance, clear and distinct labelling was obtained, either with the two antigens at separate sites, or with an overlap in distribution. In the latter situation, the sites of co-localization were marked by mixed colours, which were distinct and readily discriminated from the two basic colours.     

Analysis of antiphotobleaching reagents for use with FluoroNanogold in correlative microscopy.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence and electron microscopy. We have employed FluoroNanogold (FNG) as the detection system in these types of studies. This immunoprobe consists of a gold cluster compound to which a fluorochrome-labeled antibody is covalently linked. In these preparations, the fluorescence signal from FNG is first recorded then the gold cluster compound is subjected to a silver enhancement reaction before examination by electron microscopy. Potential complications are those associated with photochemical reactions that occur during fluorescence microscopy. We have evaluated this and some anti-photobleaching agents (i.e., 1,4-diazabicyclo[2.2.2]octane [DABCO],p-phenylenediamine [PPD], and N-propyl gallate [NPG]) for their utility with FNG in correlative microscopy. When DABCO was employed, the gold signal from FNG was dramatically diminished but the fluorescence signal was unaffected. The gold signal of DABCO-treated samples decreased to approximately 30% of that of the other samples. On the other hand, PPD and NPG did not adversely affect the FNG labeling. We recommend that either PPD or NPG be used and that DABCO be avoided as an antiphotobleaching reagent for this technique.     

The use of cell membrane stability (CMS) technique to screen for salt tolerant wheat varieties

Cell membrane stability (CMS) technique was used to screen salt tolerant (V1, V2), salt sensitive (V5) and two salt/water deficiency tolerant wheat genotypes (V3 and V4) using 100-250 mM NaCl salinity maintained in pots containing gravel and nutrient solution. The objectives were to study: (i) the reliability of CMS technique for screening wheat under high salinity, (ii) factors that impart stability and/or injury to the cell membrane, and (iii) the relationship of CMS with other physiological parameters affected by the salt stress. Generally, cellular injury increased with increasing salinity levels. In V5, it was the highest (74.2%) at 250 mM, probably due to combined effect of Na+ toxicity and low (54%) relative water content (RWC). In V1, RWC was similar to that in V5 but injury was comparatively low possibly due to low concentration of Na+. The difference between V1 and V2 was significant, either due to the highest concentration of K+ or the lowest reduction in RWC in V2. In V3 and V4, injury was the lowest at all salinity levels and was within the range of values observed earlier for drought tolerance. A significant negative correlation was detected between cellular injury and RWC for V1 and V5 but not for V3 and V4. Cellular injury also showed a significant positive correlation with Na+ and a negative correlation with K+ and grain yield (GY). It appeared that CMS technique is suitable for screening wheat under high salinity levels and for detecting differences that may arise due to cumulative effects of salinity and reduced water contents.     

High-viability lyophilized Bacille Calmette-Guérin vaccine produced by deep-culture technique.

Evidence suggests that Bacille Calmette-Guérin (BCG) vaccine for use in cancer immunotherapy should have the following characteristics: high viability which is maintained on storage; high ratio of live to dead cells; high proportion of single cells; and low content of soluble antigen. The production of a vaccine with these characteristics was accomplished by use of a deep-culture technique. The medium was modified Proskauer and Beck medium containing Tween 80 and glucose. The mass culture was grown in a Wheaton double-side-arm bottle (6 liters of medium in an 8-liter container), aerated by means of an aquarium aerator and mixed by a magnetic stirrer. The culture was incubated 7 to 9 days at 37 degrees C, concentrated 11 to 15 times by ultrafiltration, diluted with equal parts of 25% lactose, and then lyophilized. The lyophilized ampoules, stored at -70 degrees C, were cultured at intervals ranging from 3 days to 450 days, and no loss in viability was observed. The mean number of viable BCG per ml of reconstituted vaccine was 8.75 log10. The viable count was 90% of the total bacterial count. Moreover, 85% of the cells were present as single bacilli.     

The use of biotin-streptavidin systems for enhancing the parameters of immunometric analysis

The possibility of improving analytical parameters of the immunometric assay with the use of biotinylated antibodies and biotin-streptavidin complexes in comparison with the commonly known approach of direct antibody modification with 125I has been studied. Experiments have been carried out with the use of low-affinity antibodies (Kass approximately 10(9) M-1) to ferritin. The signal-to-noise ratio in the immunometric increases 2.3 times when streptavidin labeled with horse-radish peroxidase is used and 4.3 times when the preformed streptavidin + biotin-peroxidase complex is used in comparison with assay systems based on 125I-labeled antibodies. The improvement of assay parameters of immunochemical systems by means of biotin-streptavidin complexes has been found to permit the use of low-affinity antibodies as assay reagents, thus ensuring analytical parameters attaining or close to those of immunoradiometric assay systems based on high-affinity 125I-labeled antibodies (Kass approximately 10(10) M-1). As shown in this study, the following factors ensure the signal enhancement in biotin-streptoavidin systems: (a) the biotin modification of several lysin residues per IgG molecule, the optimum extent of modification being 3-4 residues per molecule; (b) mild procedure for biotinylation. In contrast to oxidative iodination, the modification of NH2 groups with biotin esters does not significantly affect their antigen-binding properties.     

The use of signalling pathway inhibitors and chromatin modifiers for enhancing pluripotency

Pluripotent embryonic stem cells have been isolated from a limited number of species. The new advances with inducing pluripotency in somatic cells have resulted in the generation of pluripotent stem cells while circumventing the need for embryos. In this review we describe the main signalling pathways involved in maintaining pluripotency and inducing differentiation. Inhibition of the signalling pathways involved in differentiation enhances the derivation and cultivation of pluripotent stem cells. Furthermore, we discuss the use of chromatin modifiers to maintain an open chromatin state which is characteristic of pluripotent stem cells, to facilitate the derivation of pluripotent cell lines.     

The monothioacetal group, a new cleavable center for cross-linking reagents.

A protein cross-linking reagent which contains a monothioacetal moiety is described. Cross-links generated using this reagent may be specifically cleaved by dilute mercuric ion at neutral pH.