Bluetongue virus: European Community inter-laboratory comparison tests to evaluate ELISA and RT-PCR detection methods.

European Community national reference laboratories participated in two inter-laboratory comparison tests in 2006 to evaluate the sensitivity and specificity of their 'in-house' ELISA and RT-PCR assays for the detection of bluetongue virus (BTV) antibodies and RNA. The first ring trial determined the ability of laboratories to detect antibodies to all 24 serotypes of BTV. The second ring trial, which included both antisera and EDTA blood samples from animals experimentally infected with the northern European strain of BTV-8, determined the ability of laboratories to detect BTV-8 antibodies and RNA, as well as the diagnostic sensitivity of the assays. A total of six C-ELISAs, six real-time RT-PCR and three conventional RT-PCR assays were used. All C-ELISAs were capable of detecting the BTV serotypes currently circulating in Europe (BTV-1, 2, 4, 8, 9 and 16), however some assays displayed inconsistencies in the detection of other serotypes, particularly BTV-19. All C-ELISAs detected BTV-8 antibodies in cattle and sheep by 21 dpi, while the majority of assays detected antibodies by 9 dpi in cattle and 8 dpi in sheep. All the RT-PCR assays were able to detect BTV-8, although the real-time assays were more sensitive compared to the conventional assays. The majority of real-time RT-PCR assays detected BTV RNA as early as 2 dpi in cattle and 3 dpi in sheep. These two ring trails provide evidence that national reference laboratories within the EC are capable of detecting BTV antibodies and RNA and provide specificity and sensitivity information on the detection methods currently available.

Investigation into the concanavalin A reactivity, fucosylation and oligosaccharide microheterogeneity of alpha 1-acid glycoprotein expressed in the sera of patients with rheumatoid arthritis.

alpha 1-Acid glycoprotein (AGP) exists as an heterogeneous population of glycosylated variants (glycoforms) in plasma. The concentration of AGP increases some 2-5 fold in certain pathophysiological states exemplified by the chronic inflammatory disease, rheumatoid arthritis (RA). Moreover, the expressed glycosylation pattern alters in such conditions, indicating functional significance that is likely to be related to the oligosaccharide heterogeneity. We have investigated the heterogeneity of AGP glycosylation using the technique of high pH anion-exchange chromatography (HPAEC). AGP was isolated from the blood of RA sufferers, partially separated by Concanavalin A (Con A) affinity chromatography into bound and non-bound fractions and was enzymatically deglycosylated. Chromatography on the pellicular HPAE resin at pH 13 separated the released oligosaccharides and allowed a comparison of profiles in terms of branching and fucosylation. Results demonstrate an abnormal RA AGP glycosylation, with a tendency towards tri- and tetra-antennary oligosaccharides and enhanced fucosylation, in addition to the possible existence of penta-sialylated RA AGP glycoforms.

Chromatographic investigation of the glycosylation pattern of alpha-1-acid glycoprotein secreted by the HepG2 cell line; a putative model for inflammation?

In certain pathophysiological conditions, such as rheumatoid arthritis, there are alterations in the glycosylation pattern of the acute phase protein, alpha-1-acid glycoprotein (AGP). These changes are likely to be functionally significant, however, verification of the latter role requires a system which reflects in vivo glycosylation changes in AGP and also produces sufficient quantities of the protein for further study. The human hepatoma cell line HepG2 is documented as displaying a shift in the glycosylation pattern of glycoproteins from normal state to acute phase after stimulation with inflammatory mediators. We have isolated AGP from the culture medium of HepG2 cells both before and after stimulation with a cytokine preparation and analysed the glycosylation pattern of each preparation, after enzymatic release, by high pH anion-exchange chromatography. Before stimulation, the glycosylated population was similar to a profile of AGP isolated from normal plasma; however, cytokine stimulation resulted in a shift to a profile which was consistent with that of AGP from a rheumatoid arthritis sufferer. Thus a HepG2 cell culture system is capable of being a crude model of the changes in glycosylation of acute phase proteins although it has a tendency to produce oligosaccharide chains which are not fully sialylated.

Heterogeneity of alpha 1-acid glycoprotein in rheumatoid arthritis.

alpha 1-Acid glycoprotein (AGP) or orosomucoid is a major serum glycoprotein, of unknown physiological function, which is classified as one of the positive acute phase reactants since its plasma concentration becomes elevated two- to five-fold in certain disease states. Additionally, the proportions and identities of the five asparaginyl-linked complex oligosaccharide chains are altered during several physiological and pathological conditions, which may be functionally significant. The key to studying the structural heterogeneity of AGP is to develop a procedure that will isolate AGP without structural degradation. We have developed a method for the purification of AGP, using procedures unlikely to damage the glycoprotein structure, which was utilised to isolate AGP from samples of normal and rheumatoid plasma. The effectiveness of the purification procedure was examined by enzymatically deglycosylating each sample of AGP and separating the released oligosaccharides by chromatography on a pellicular high-performance anion-exchange (HPAE) resin at pH 13. The analytical profile for normal AGP was consistent with that previously reported thus indicating that the purification procedure did not denature the oligosaccharide chains of AGP. Additionally, there was a noticeable difference between the profiles for AGP from normal and rheumatoid plasma.