Antigenic features of protein carriers commonly used in immunisation trials.

An aluminium hydroxide adjuvant induced a more elevated and rapid immune responses against short peptides conjugated to the Keyhole Lympet Hemocyanin carrier than immuneasy adjuvant. Furthermore, since carrier proteins may compete with the fused or chemically linked polypeptides in eliciting antigen-specific immune response, we classified the immunogenicity of the most common carrier proteins used in molecular biology for antigen expression and mouse immunisation. The disulfide isomerase protein A gave a carrier with the lowest immunogenicity whilst disulfide isomerase protein C gave the highest immunogenicity and therefore should be avoided as a fusion partner. Using this protein as a model, we identified and located the immunodominant epitopes along its sequence. These results now enable the combination of carrier and immunisation conditions to be optimized.

Optimization of trap color for emerald ash borer (Coleoptera: Buprestidae).

Field assays were performed to determine the optimal color for Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) traps. Previous studies have found that more A. planipennis are caught on purple or green traps than traps of other colors. In three studies, we evaluated various shades of purple, wavelengths of green (500-570 nm), and greens of different reflectance (from 9 to 66%). In all tests, traps of corrugated plastic in standard, commercially available purple (currently used to survey A. planipennis) and a customized green color were used as bases for comparison. Among purple traps, a paint color previously shown to be generally attractive to buprestids caught significantly more A. planipennis adults than traps coated with paints containing more blue or red, or traps constructed of the standard purple plastic. Among traps with maximum reflectance at varying green wavelengths, those ranging in wavelength from 525 to 540 nm caught significantly more adult A. planipennis than traps of other wavelengths. In the 530-540 nm range of the electromagnetic spectrum, there was no significant difference among traps in the 23-66% reflectance range, but traps painted with a peak reflectance of 49% caught more beetles than purple or the custom green plastic traps. Male to female ratio was highest on green traps.

Laboratory and field response of the emerald ash borer (Coleoptera: Buprestidae), to selected regions of the electromagnetic spectrum.

Retinal sensitivity of Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) was examined with an aim to improve trap efficacy for the beetle. Electroretinogram (ERG) recordings from dark-adapted compound eyes of male and female were measured at different wavelengths across the spectrum ranging from 300 to 700 nm. The spectral sensitivity curves revealed peaks in the UV (340 nm), the violet/purple (420-430 nm), blue (460 nm), and green (540-560 nm) regions of the spectrum. Females were sensitive to red regions of the spectrum (640-670 nm), whereas males were not. A spectrophotometer was used to measure the wavelength and reflectance for ash foliage, purple corrugated plastic traps, as well as the elytra and abdomen of adult A. planipennis. Traps were painted using colors based on ERG and spectrophotometer measurements and compared with purple corrugated plastic traps currently used by the USDA-APHIS-PPQ-EAB National Survey. In a field assay conducted along the edges of several A. planipennis-infested ash stands, there were no significant differences in trap catch among green, red, or purple treatments. Dark blue traps caught significantly fewer A. planipennis than red, light green, or dark purple traps. In a second assay where purple and green treatments were placed in the mid canopy of ash trees (approximately 13 m in height), trap catch was significantly higher on green treatments. We hypothesize that when placed in the mid-canopy, green traps constitute a foliage-type stimulus that elicits food-seeking and/or host seeking behavior by A. planipennis.

Development of a host-based semiochemical lure for trapping emerald ash borer Agrilus planipennis (Coleoptera: Buprestidae).

Bark volatiles from green ash Fraxinus pennsylvanica were tested for electrophysiological activity by Agrilus planipennis using gas chromatographic-electroantennographic detection (GC-EAD) and for behavioral activity using baited purple traps in Michigan. GC-EAD analysis of the headspace volatiles of bark tissue samples from 0- and 24-h-old fully girdled (stressed) ash trees showed that the latter had elevated sesquiterpene levels. Six of the elevated compounds consistently elicited antennal responses by both male and female A. planipennis. Five of the antennally active compounds were identified as alpha-cubebene, alpha-copaene, 7-epi-sesquithujene, trans-beta-caryophyllene, and alpha-humulene (alpha-caryophyllene). The sixth EAD-active compound remains unidentified. We monitored capture of adult A. planipennis on traps baited with several combinations of ash tree volatiles. Treatments included two natural oil distillates (Manuka and Phoebe oil) that were found to contain, respectively, high concentrations of four and five of the six antennally active ash bark volatiles. A four-component leaf lure developed by the USDA Forest Service and Canadian Forest Service was also tested. In three separate field studies, Manuka oil-baited traps caught significantly more adult beetles than unbaited traps. Lures designed to release 5, 50, and 500 mg of Manuka oil per day all caught more insects than unbaited traps. In a field test comparing and combining Phoebe oil with Manuka oil, Phoebe oil-baited traps caught significantly more beetles than either Manuka oil-baited traps or unbaited traps. We hypothesize that the improved attractancy of Phoebe oil to A. planipennis over Manuka oil is caused by the presence of the antennally active sesquiterpene, 7-epi-sesquithujene.

Influence of trap placement and design on capture of the emerald ash borer (Coleoptera: Buprestidae).

The key to an effective pest management program for the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera Buprestidae), is a survey program equipped with tools for detecting and delimiting populations. We studied the effects of trap design, color, and placement on the efficacy of sticky traps for capturing the emerald ash borer. There were significant differences in trap catch along a transect gradient from wooded to open field conditions, with most beetles being caught along the edge, or in open fields, 15-25 m outside an ash (Fraxinus spp. L.) (Oleaceae) woodlot. Greater emerald ash borer catch occurred on purple traps than on red or white traps. Traps placed in the mid-canopy of ash trees (13 m) caught significantly more beetles than those placed at ground level. We also describe a new trap design, a three-sided prism trap, which is relatively easy to assemble and deploy.

Unrip is a component of SMN complexes active in snRNP assembly.

A macromolecular complex containing survival of motor neurons (SMN), the spinal muscular atrophy protein, and Gemin2-7 interacts with Sm proteins and snRNAs to carry out the assembly of these components into spliceosomal small nuclear ribonucleoproteins (snRNPs). Here we report the characterization of unr-interacting protein (unrip), a GH-WD protein of unknown function, as a component of the SMN complex that interacts directly with Gemin6 and Gemin7. Unrip also binds a subset of Sm proteins, and unrip-containing SMN complexes are necessary and sufficient to mediate the assembly of spliceosomal snRNPs. These results demonstrate that unrip functions in the pathway of snRNP biogenesis and is a marker of cellular SMN complexes active in snRNP assembly.

Plasmodium falciparum rosetting epitopes converge in the SD3-loop of PfEMP1-DBL1α.

The ability of Plasmodium falciparum parasitized RBC (pRBC) to form rosettes with normal RBC is linked to the virulence of the parasite and RBC polymorphisms that weaken rosetting confer protection against severe malaria. The adhesin PfEMP1 mediates the binding and specific antibodies prevent sequestration in the micro-vasculature, as seen in animal models. Here we demonstrate that epitopes targeted by rosette disrupting antibodies converge in the loop of subdomain 3 (SD3) which connects the h6 and h7 α-helices of PfEMP1-DBL1α. Both monoclonal antibodies and polyclonal IgG, that bound to epitopes in the SD3-loop, stained the surface of pRBC, disrupted rosettes and blocked direct binding of recombinant NTS-DBL1α to RBC. Depletion of polyclonal IgG raised to NTS-DBL1α on a SD3 loop-peptide removed the anti-rosetting activity. Immunizations with recombinant subdomain 1 (SD1), subdomain 2 (SD2) or SD3 all generated antibodies reacting with the pRBC-surface but only the sera of animals immunized with SD3 disrupted rosettes. SD3-sequences were found to segregate phylogenetically into two groups (A/B). Group A included rosetting sequences that were associated with two cysteine-residues present in the SD2-domain while group B included those with three or more cysteines. Our results suggest that the SD3 loop of PfEMP1-DBL1α is an important target of anti-rosetting activity, clarifying the molecular basis of the development of variant-specific rosette disrupting antibodies.

Comparison and critical analysis of robotized technology for monoclonal antibody high-throughput production.

We have previously demonstrated how to transform the conventional method of hybridoma production and screening into a fast, high-throughput technology. Nevertheless, there were still open questions related to automated procedures and immunization protocols that we address now by comparing the hybridoma production work-flow in automated and manually executed processes. In addition, since the animals' antibody responses to single or multiple antigen challenge affect monoclonal antibody throughput, different immunization and fusion strategies were tested. Specifically, the results obtained with multiplexing (multiple target antigens injected into a single animal) and single antigen immunization followed by splenocyte pooling immediately before fusion were compared with conventional methods. The results presented here demonstrate that the optimal protocol consists of automated somatic-cell fusion and hybridoma dilution followed by manual plating of hybridoma cells. Additionally, more specific and productive hybridoma clones were obtained with multiplexed immunization in a single animal with respect to the splenocyte pooling from single antigen immunized animals. However, in terms of overall antibody yield, the conventional method consisting of single immunization for each single animal assured ten times more specific hybridoma cell lines than the strategy based on the multiple antigen immunization followed by separate fusion step. In conclusion, the most productive approach for recovering a large number of suitable antibodies relies on single antigen immunization followed by automated fusion and dilution steps and manual plating.

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome.

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Gemin8 is a novel component of the survival motor neuron complex and functions in small nuclear ribonucleoprotein assembly.

The survival motor neuron (SMN) protein is the product of the spinal muscular atrophy disease gene. SMN and Gemin2-7 proteins form a large macromolecular complex that localizes in the cytoplasm as well as in the nucleoplasm and in nuclear Gems. The SMN complex interacts with several additional proteins and likely functions in multiple cellular pathways. In the cytoplasm, a subset of SMN complexes containing unrip and Sm proteins mediates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Here, by mass spectrometry analysis of SMN complexes purified from HeLa cells, we identified a novel protein that is evolutionarily conserved in metazoans, and we named it Gemin8. Co-immunoprecipitation and immunolocalization experiments demonstrated that Gemin8 is associated with the SMN complex and is localized in the cytoplasm and in the nucleus, where it is highly concentrated in Gems. Gemin8 interacts directly with the Gemin6-Gemin7 heterodimer and, together with unrip, these proteins form a heteromeric subunit of the SMN complex. Gemin8 is also associated with Sm proteins, and Gemin8-containing SMN complexes are competent to carry out snRNP assembly. Importantly, RNA interference experiments indicate that Gemin8 knock-down impairs snRNP assembly, and Gemin8 expression is down-regulated in cells with low levels of SMN. These results demonstrate that Gemin8 is a novel integral component of the SMN complex and extend the repertoire of cellular proteins involved in the pathway of snRNP biogenesis.

B cell receptors in TCL1 transgenic mice resemble those of aggressive, treatment-resistant human chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (B-CLL) is a clonal overgrowth of CD5(+) B lymphocytes. In this disease, the B cell antigen receptor (BCR) is intimately linked to disease severity, because patients with BCRs, comprised of unmutated V(H) genes, follow a much more aggressive course. This and related observations suggest that B-CLL derives from a B cell subset comprised of restricted BCR structural diversity and that antigen-selection and drive are major factors promoting the disease. Nevertheless, the initiating event(s) that lead to the development of B-CLL are still unclear, in part because of the lack of an animal model that spontaneously evolves the molecular abnormalities that occur in the human disease. Because overexpression of the TCL1 gene in murine B cells leads to a CD5(+) B cell lymphoproliferative disorder with many of the features of human B-CLL, we studied leukemias emerging in these mice to examine the extent to which their BCRs resemble those in B-CLL. Our data indicate that the immunoglobulin heavy and light chain rearrangements in TCL1 mice display minimal levels of somatic mutations and exhibit several molecular features found in the human disease. Like human B-CLL, TCL1 leukemic rearrangements from different mice can be very similar structurally and closely resemble autoantibodies and antibodies reactive with microbial antigens. Antigen-binding analyses confirm that selected TCL1 clones react with glycerophospholipid, lipoprotein, and polysaccharides that can be autoantigens and be expressed by microbes. This (auto)antigen-driven mouse model reliably captures the BCR characteristics of aggressive, treatment-resistant human B-CLL.

High throughput production of mouse monoclonal antibodies using antigen microarrays.

Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification. Monoclonal antibodies were raised to different targets in single batch runs of 6-10 wk using multiplexed immunisations, automated fusion and cell-culture, and a novel antigen-coated microarray-screening assay. In a large-scale experiment, where eight mice were immunized with ten antigens each, we generated monoclonal antibodies against 68 of the targets (85%), within 6 wk of the primary immunization.