Optimisation of the enzyme-linked lectin assay for enhanced glycoprotein and glycoconjugate analysis

Lectin’s are proteins capable of recognising and binding to specific oligosaccharide tructures found on glycoproteins and other biomoloecules. As such they have found tility for glycoanalytical applications. One common difficulty encountered in the pplication of these proteins, particularly in multi-well plate assay formats known as Enzyme Linked Lectin Assays (ELLA’s), is in finding appropriate blocking solutions to prevent non-specific binding with plate surfaces. Many commonly used blocking agents contain carbohydrates and generate significant background signals in ELLA’s, limiting the utility of the assay. In this study we examined the suitability of a range of blocking reagents, including rotein based, synthetic and commercially available carbohydrate free blocking eagents, for ELLA applications. Each blocking reagent was assessed against a panel f 19 commercially available biotinylated lectins exhibiting diverse structures and arbohydrate specificities. We identified the synthetic polymer Polyvinyl Alcohol PVA) as the best global blocking agent for performing ELLA’s. We ultimately present n ELLA methodology facilitating broad spectrum lectin analysis of glycoconjugates nd extending the utility of the ELLA

The relationship between anti-HPV-16 IgG seropositivity and cancer of the cervix, anogenital organs, oral cavity and pharynx, oesophagus and prostate in a black South African population

BACKGROUND: Human papillomavirus type 16 (HPV-16) infection is an important cause of cervical cancer, other anogenital cancers and, possibly, some oral and pharyngeal cancers. The association of HPV-16 with oesophageal and with prostate cancers has not been firmly established. METHODS: We analysed sera from 3,757 HIV seronegative black South Africans using an anti-HPV IgG enzyme-linked immunosorbent assay (ELISA). The subjects were recruited from 1995 to 2000 as part of an ongoing cancer case control study. Cases were patients with newly diagnosed cancers of the cervix (n = 946), other anogenital organs (n = 80), the oral cavity and pharynx (n = 102), the oesophagus (n = 369) or the prostate (n = 205). The comparison group consisted of 2,055 age and sex-matched patients randomly selected from the same data base, diagnosed at the same hospitals, but with a vascular disease or with a cancer unrelated to HPV infection. Subjects' sera were randomly and blindly allocated onto ELISA plates. Optical density (OD) levels of anti-HPV-16 IgG of > 0.45 and ≥ 0.767 were taken to be cut-offs for negative, medium and high antibody levels. RESULTS: After adjustment for potential confounders, cancer types that showed a statistically significant association with increased anti-HPV-16 IgG antibody (Ab) levels were cancer of the cervix (OR for medium Ab levels = 1.6, and for high = 2.4, p < 0.0001), cancers of other anogenital organs (OR for medium or high Ab levels = 2.5, p = 0.002), and cancer of the oesophagus (OR for medium Ab = 1.3, and high Ab levels = 1.6 p = 0.002). Cancers of the oral cavity and pharynx showed a borderline significant association in the unadjusted model (p = 0.05) but after adjustment for confounding the trend in relation to Ab levels was positive but not statistically significant (OR for medium Ab = 1.1, and high Ab = 1.5 p = 0.13). Prostate cancer was not associated with HPV-16 seropositivity (OR for medium Ab level = 1.4, and for high Ab level = 1.3, p = 0.3). CONCLUSION: If there is indeed an association between HPV-16 and oesophageal and possibly also some oral cavity and pharyngeal cancers, then emerging HPV vaccines may also reduce, at least in part, the incidence of these leading cancer types

Covalent immobilisation of antibodies in Teflon-FEP microfluidic devices for sensitive quantification of clinically relevant protein biomarkers

This study reports for the first time sensitive colorimetric and fluorescence detection of clinically relevant protein biomarkers by sandwich immunoassays using covalent immobilisation of antibodies onto the fluoropolymer surface inside Teflon®-FEP microfluidic devices. Teflon®-FEP has outstanding optical transparency ideal for high-sensitivity colorimetric and fluorescence bioassays, however this thermoplastic is regarded as chemically inert and very hydrophobic. Covalent immobilisation can offer benefits over passive adsorption to plastic surfaces by allowing better control over antibody density, orientation and analyte binding capacity, and so we tested a range of different and novel covalent immobilisation strategies. We first functionalised the inner surface of a 10-bore, 200 µm internal diameter FEP microcapillary film with high-molecular weight polyvinyl alcohol (PVOH) without changing the outstanding optical transparency of the device delivered by the matched refractive index of FEP and water. Glutaraldehyde immobilisation was compared with use of photoactivated linkers and NHS-ester crosslinkers for covalently immobilising capture antibodies onto PVOH. Three clinically relevant sandwich ELISAs were tested, against the cytokine IL-1ß, the myocardial infarct marker cardiac troponin I (cTnI), and the chronic heart failure marker brain natriuretic peptide (BNP). Overall, glutaraldehyde immobilisation was effective for BNP assays, but yielded unacceptable background for IL-1ß and cTnI assays caused by direct binding of biotinylated detection antibody to the modified PVOH surface. We found NHS-ester groups reacted with APTES-treated PVOH coated fluoropolymer. This facilitated a novel method for capture antibody immobilisation onto fluoropolymer devices using a bifunctional NHS-maleimide crosslinker. The density of covalently immobilised capture antibodies achieved using PVOH/APTES/NHS/Maleimide approached levels seen with passive adsorption, and sensitive and quantitative assay performance was achieved using this method. Overall, PVOH coating provided an excellent surface for controlled covalent antibody immobilisation onto Teflon®-FEP for performing high-sensitivity immunoassays

La nuova Russia. Dibattito culturale e modello di società in costruzione

Una radiografia del dibattito culturale della Russia che illustra i tanti problemi con cui si deve confrontare la costruzione di una nuova società. Modernizzazione, tradizionalismo, nuovo corporativismo, privatizzazioni, nazionalismo e religione sono solo alcuni degli elementi che compongono un quadro ricco di conflittualità e foriero di grandi trasformazioni.- Indice #5- Radiografia del dibattito culturale: modernizzazione e tradizionalismo nello scontro tra gli “addetti ai lavori”, Ilja Levin #9- Partiti e società: evoluzione e prospettive della differenziazione politica ed ideologica nella Russia postsovietica, Kirill Kholodkovski #29- Dal “collettivismo” all’individualismo: l’uomo nella società deistituzionalizzata, Guerman Diliguenski #45- Gruppi direttivi regionali: trasformazione dei meccanismi organizzativi e relazionali del potere, Mikhail Afanasjev #63- L’”oligarchia” e la crisi in atto nel postcomunismo russo, Alexeij Zudin #81- Il nuovo corporativismo russo all’interno del contesto globale: il ruolo in via di cambiamento dell’élite settoriale, Sergei Peregudov #107- La formazione di nuovi mercati e lo Stato nella Russia postcomunista, Vadim Radaev #133- Stabilizzazione macroeconomica e mutamenti strutturali nell’economia nazionale russa: deindustrializzazione o sfondamento verso una società postindustriale?, Vladimir Mau e Irina Starodubrovskaja #163- Dopo la privatizzazione: alla ricerca di un modello ottimale di società, Viktor Studentsov #187- Fondamenta e principi dello Stato accentratore in Russia, Igor Pantin #217- Nazionalismo liberale o idea imperiale? La consapevolezza degli interessi e degli orientamenti nazionali in Russia, Vladimir Kolossov #233- Il primo quinquennio della repubblica di dicembre in Russia: come si costruiscono gli istituti e si strutturano gli interessi, Petr Fedossov #257- Lo stato attuale e le prospettive della Chiesa ortodossa in Russia, P. Innokentij Pavlov #272- La nuova influenza dei valori religiosi sull’intellighenzia russa (tendenze attuali), Andrei B. Zubov #295- La religiosità postsovietica: dall’eclettismo religioso alle fedi nazionali, Sergei Filatov #32

More men than women make mucosal IgA antibodies to Human papillomavirus type 16 (HPV-16) and HPV-18: a study of oral HPV and oral HPV antibodies in a normal healthy population

BACKGROUND: We have previously shown the high prevalence of oral anti-human papillomavirus type 16 (HPV-16) antibodies in women with HPV-associated cervical neoplasia. It was postulated that the HPV antibodies were initiated after HPV antigenic stimulation at the cervix via the common mucosal immune system. The present study aimed to further evaluate the effectiveness of oral fluid testing for detecting the mucosal humoral response to HPV infection and to advance our limited understanding of the immune response to HPV. METHODS: The prevalence of oral HPV infection and oral antibodies to HPV types 16, 18 and 11 was determined in a normal, healthy population of children, adolescents and adults, both male and female, attending a dental clinic. HPV types in buccal cells were determined by DNA sequencing. Oral fluid was collected from the gingival crevice of the mouth by the OraSure method. HPV-16, HPV-18 and HPV-11 antibodies in oral fluid were detected by virus-like particle-based enzyme-linked immunosorbent assay. As a reference group 44 women with cervical neoplasia were included in the study. RESULTS: Oral HPV infection was highest in children (9/114, 7.9%), followed by adolescents (4/78, 5.1%), and lowest in normal adults (4/116, 3.5%). The predominant HPV type found was HPV-13 (7/22, 31.8%) followed by HPV-32 (5/22, 22.7%). The prevalence of oral antibodies to HPV-16, HPV-18 and HPV-11 was low in children and increased substantially in adolescents and normal adults. Oral HPV-16 IgA was significantly more prevalent in women with cervical neoplasia (30/44, 68.2%) than the women from the dental clinic (18/69, 26.1% P = 0.0001). Significantly more adult men than women displayed oral HPV-16 IgA (30/47 compared with 18/69, OR 5.0, 95% CI 2.09–12.1, P < 0.001) and HPV-18 IgA (17/47 compared with 13/69, OR 2.4, 95% CI 0.97–6.2, P = 0.04). CONCLUSION: The increased prevalence of oral HPV antibodies in adolescent individuals compared with children was attributed to the onset of sexual activity. The increased prevalence of oral anti-HPV IgA in men compared with women was noteworthy considering reportedly fewer men than women make serum antibodies, and warrants further investigation

Non-material factors in the works of the German historical school of Economics

The German historical school of economics (GHSE) was one of only a few original economic phenomena to have left a profound and indelible trace in economic science. Basing themselves on the school’s special methodology and its perspective on economic processes, adherents of the GHSE formulated many ideas and approaches that were important for their time. In this way, the GHSE issued a challenge to the then-dominant British classical political economy, with its abstract and speculative constructs and exaggeration of the role of a deductive as opposed to an inductive method of analysis. In the concepts of the GHSE, a special role was played by non-material factors of production, and by the fact that the subject was rooted in the system of the national economy. Such phenomena as morality, law, culture in the broad sense of the word, the spiritual principles (Geist) of economic activity, and similar aspects of economic activity that, it seemed, were not clearly defined or understood were ignored by classical political economy and left outside the bounds of the dominant principles of politico-economic analysis. Meanwhile, it was precisely these that made up the core of the concept, developed by the German economists, of the national historical human being, of the motives behind his or her interaction with those round about, and of the principles shaping the national economy. In our own time, many of the ideas of the GHSE have been taken up by the neoinstitutionalists and by economists operating outside the framework of the neoclassical mainstream