Application of cover-free codes and combinatorial designs to two-stage testing

AbstractWe study combinatorial and probabilistic properties of cover-free codes and block designs which are useful for their efficient application as the first stage of two-stage group testing procedures. Particular attention is paid to these procedures because of their importance in such applications as monoclonal antibody generation and cDNA library screening

Application of phage display to high throughput antibody generation and characterization.

We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

T-SP1: a novel serine protease-like protein predominantly expressed in testis

Here, we describe a novel member in the group of membrane-anchored chymotrypsin (S1)-like serine proteases, namely testis serine protease 1 (T-SP1), as it is principally expressed in testis tissue. The human T-SP1 gene encompasses 28.7 kb on the short arm of chromosome 8 and consists of seven exons. Rapid amplification of cDNA ends ( RACE) experiments revealed that due to alternative splicing three different variants (T-SP1/1, -2, -3) are detectable in testis tissue displaying pronounced heterogeneity at their 3'-end. T-SP1/1 consists of an 18 amino acid signal peptide and of a 49 amino acid propeptide. The following domain with the catalytic triad of His(108), Asp(156), and Ser(250) shares sequence identities of 42% and 40% with the blood coagulation factor XI and plasma kallikrein, respectively. Only T-SP1/1 contains a hydrophobic part at the C-terminus, which provides the basis for cell membrane anchoring. Using a newly generated polyclonal anti-T-SP1 antibody, expression of the T-SP1 protein was found in the Leydig and Sertoli cells of the testis and in the epithelial cells of the ductuli efferentes. Notably, T-SP1 protein was also detectable in prostate cancer and in some ovarian cancer tissues, indicating tumor-related synthesis of T-SP1 beyond testis tissue

Drosophila Bruce Can Potently Suppress Rpr- and Grim-Dependent but Not Hid-Dependent Cell Death

Bruce is a large protein (530 kDa) that contains an N-terminal baculovirus IAP repeat (BIR) and a C-terminal ubiquitin conjugation domain (E2) 1, 2. BRUCE upregulation occurs in some cancers and contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs [2]. However, it is still unknown whether Bruce inhibits apoptosis directly or instead plays some other more indirect role in mediating chemoresistance, perhaps by promoting drug export, decreasing the efficacy of DNA damage-dependent cell death signaling, or by promoting DNA repair. Here, we demonstrate, using gain-of-function and deletion alleles, that Drosophila Bruce (dBruce) can potently inhibit cell death induced by the essential Drosophila cell death activators Reaper (Rpr) and Grim but not Head involution defective (Hid). The dBruce BIR domain is not sufficient for this activity, and the E2 domain is likely required. dBruce does not promote Rpr or Grim degradation directly, but its antiapoptotic actions do require that their N termini, required for interaction with DIAP1 BIR2, be intact. dBruce does not block the activity of the apical cell death caspase Dronc or the proapoptotic Bcl-2 family member Debcl/Drob-1/dBorg-1/Dbok. Together, these results argue that dBruce can regulate cell death at a novel point

Actinidin : the predominant protease in kiwifruit : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Food technology at Massey University, Manawatū, New Zealand

Kiwifruit protein (actinidin) has been widely known as a protease. Kiwifruit protein has the potential of utilization in food industry as an enzyme that aids food digestion. In this project, the soluble kiwifruit proteins were extracted from fresh Hayward and SunGold kiwifruit. Soluble kiwifruit proteins were analysed by the Hartree-Lowry method, SDS-PAGE, enzyme activity determination, ion-exchange chromatography and mass spectrometry. Anti-actinidin antibodies were raised by the injection of purified actinidin into rabbits. The main soluble kiwifruit protein was recognized by anti-actinidin antibodies using Western blot. Moreover, the effects of post-harvest storage on protein content, total enzyme activity and specific enzyme activity were investigated. Comparable studies on both Hayward and SunGold kiwifruit were also carried out in this project. The results showed that Hayward and SunGold kiwifruit had a similar protein content. However, the total enzyme activity of Hayward kiwifruit was about 8 times higher than that of SunGold kiwifruit. The protein with enzyme activity (active actinidin) had a molecular weight of about 27 kDa according to SDS-PAGE and was one of main soluble proteins in Hayward and SunGold kiwifruit. This protease was purified from fresh kiwifruit by anion-exchange chromatography. A polyclonal antibody against actinidin was successfully generated in a rabbit using purified actinidin. Protein with a molecular weight of 27 kDa was recognized by the anti-actinidin antibodies. Post-harvest storage at 1 °C for up to 12 weeks significantly increased the total and specific enzyme activities of SunGold kiwifruit (P<0.05). By contrast, the total and specific enzyme activities of Hayward kiwifruit had a significant decrease after 16 weeks’ storage (P<0.05). Hayward kiwifruit had no significant changes in protein content after storage (P<0.05) while the protein content of SunGold kiwifruit fluctuated in a range from 5.04 to 5.84 mg/mL during post-harvest storage. This study may help to understand the nature of kiwifruit proteins with enzyme activity, which contributes to a full understanding of the health benefits of kiwifruit

Fructan biosynthesis in Lolium perenne : tissue, cultivar and temperature effects on gene expression and protein accumulation profiles : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand

Cultivars of Lolium perenne with high concentrations of water soluble carbohydrates (WSCs) offer opportunities to mitigate greenhouse gas emissions (nitrous oxides) from grazed pastures and improve meat and milk production in livestock. Our previous studies demonstrated that fructan accumulation in the blades of high W SC grasses involves a strong gene x environment interaction. To identify the temperature effects on the expression of high sugar trait in the high sugar cultivars. we conducted a pot trial in climate chambers with temperature regimes set at10/10, 20/10 and 20/20°C (day/night), respectively. Water soluble carbohydrate concentrations, the expression of the key genes and proteins: l-SST (sucrose: sucrose l-fructosyltransferase), l-FFT (fructan: fructan l-fruclosyltransferase), 6G-FFT (fructan: fructan 6G-fructosyltransferase) and l-FEH l-fructan exohydrolases) involved in the fructan biosynthetic pathway of L. perenne were compared in blades and sheaths of three selected high sugar cultivars (P, A and H) and a common cultivar (F) grown under the three temperature regimes. We found that amongst the selected 3 high sugar cultivars, high molecular weight (HMW) WSC content was significantly higher in P and A cultivars, regardless of the temperature regimes. As expected, sheaths contained significantly higher concentrations of HMW WSCs (fructans) compared to leaf blades. The highest WSC contents in both leaf and sheath tissues accumulated at 10/10°C while the lowest accumulated at 20/20°C. Gene expression profiles demonstrated that all four genes studied were more significantly expressed in sheaths compared to blades, and the expression levels were highly correlated with fructan accumulation in this tissue. Low temperature resulted in significant up-regulation of l-SST in sheaths, but not in blades. l-FFT was highly expressed in blades of A and P cultivars. Unexpectedly. 6G-FFT was expressed more significantly in the control F cultivar. but not in the high sugar cultivar P. Protein expression profiles showed that l -SST protein accumulated to high levels in sheaths, whereas protein levels of l-FFT and l-FEH were higher in blades. l-SST protein levels in both blades and sheaths generally increased in plants grown at low temperatures, whereas l-FFT protein was not affected by low temperatures in blades and sheaths, furthermore, in both tissues there was no consistent effect observed between the different cultivars and temperature regimes on l-FEH protein levels

Synthetic studies towards catalytic antibody generation

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN017302 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Role and immunomodulatory profile of histamine receptors by H1 and H2 antagonists

The present study was designed to delineate the immunomodulatory role of histamine receptors (H1- and H2-) on induction of antibody response to sheep red blood cells (SRBC), as well as the antibody generation profile, in rabbit system, systemically. The rabbits in two groups received pheniramine (H1-receptor antagonist) and ranitidine (H2-receptor antagonist), respectively, via intramuscular route and were immunized with SRBC intravenously to evaluate suppression or enhancement of antibody responses in sem. A third, control group, received vehicle and were immunized in a similar manner. Histamine released from effector cells (mast cells and basophils) _in vivo_ during inflammatory reactions could influence a detectable antibody response to SRBC as early as day 7-postimmunization (post-I), which lasted until day 28- post-I. Pheniramine-treated rabbits had significantly (*Pa &#x2264; 0.05 and **Pa &#x2264; 0.01) more suppressed total serum antibody (IgM + IgG) to SRBC as compared to ranitidine-treated ad cotrol rabbits, while ranitidine-treated rabbits showed different pattern (suppressed or enhanced) during the whole study period. Ranitidine suppressed total antibody level at days 7- and 14- post-I, and enhanced at days 21- and 28- post-I. IgM suppression at day 7- and enhancement at days 14-, 21- and 28- post-I, while IgG suppression during whole study period, as compared to control group was significant (*Pa &#x2264; 0.05 and **Pa &#x2264; 0.01) as assessed by direct hemagglutination assay* ad whole SBC-ELISA method**. Here we report that histamine receptor type 2 (H2R)-antagonists have a dominant role on immunosuppression and in immunoregulation of humoral immune responses. Histamine receptor type 2 (H2R)-antagonists are mainly involved in B cell differentiation and proliferation over histamine receptor type 1 (H1R)-antagonists

Ready Access to Proquinazid Haptens via Cross-Coupling Chemistry for Antibody Generation and Immunoassay Development

17 pages, 4 tables, 5 figures.-- Publisher's PDFBioconjugate preparation is a fundamental step for antibody generation and immunoassay development to small chemical compounds. For analytical targets holding in their structure an aryl halogen atom, cross-coupling reactions may be a simple and efficient way to obtain functionalized derivatives; thus offering great potential to elicit robust and selective immune responses after being coupled to immunogenic carrier proteins. However, substitution of the halogen atom by an aliphatic chain might eventually compromise the affinity and specificity of the resulting antibodies. In order to address this issue, proquinazid, a new-generation fungicide with outstanding performance, was chosen as model analyte. Two functionalized derivatives differing in spacer arm rigidity were synthesized by Sonogashira cross-coupling chemistry. These haptens were covalently coupled to bovine serum albumin and the resulting immunoconjugates were employed for rabbit vaccination. Antibodies were tested for proquinazid recognition by direct and indirect competitive immunoassay, and IC50 values in the low nanomolar range were found, thus demonstrating the suitability of this straightforward synthetic strategy for the generation of immunoreagents to compounds bearing an aryl halide. Following antibody characterization, competitive immunoassays were developed and employed to determine proquinazid residues in grape musts, and their analytical performance was satisfactorily validated by comparison with GC–MS. Besides having described the development of the first immunochemical method for proquinazid analysis, an efficient functionalization approach for analytes comprising aryl halides is reported.Peer reviewe