DEVELOPMENT OF A SENSITIVE AND SPECIFIC TIME-RESOLVED FLUOROMETRIC IMMUNOASSAY FOR THE BOVINE ACUTE-PHASE PROTEIN HAPTOGLOBIN (HP)

Haptoglobin (Hp) is recognised as a major acute phase protein in the bovidae and its presence in serum is used as an indicator of inflammation. A mouse monoclonal antibody (1D9) specific for bovine Hp was labelled with a lanthanide (Eu) chelate and used to develop a competitive immunoassay. This competitive immunoassay allowed direct measurement of Hp in serum and was validated for intra- and interassay coefficients of variation (below 8%). Cross-reactivity with other serum proteins was measured (less than 0.1%) and limits of detection for Hp in serum were established for adult male (0.344 mu g/ml) and adult female cattle (1.589 mu g/ml). The immunoassay was compared with an established haptoglobin-haemoglobin binding assay.

Cell-specific processing of chromogranin A in endocrine cells of the rat stomach

The rat stomach is rich in endocrine cells. The acid-producing (oxyntic) mucosa contains ECL cells, A-like cells, and somatostatin (D) cells, and the antrum harbours gastrin (G) cells, enterochromaffin (EC) cells and D cells. Although chromogranin A (CgA) occurs in all these cells, its processing appears to differ from one cell type to another. Eleven antisera generated to different regions of rat CgA, two antisera generated to a human (h) CgA sequences, and one to a bovine Ib) CgA sequence, respectively, were employed together with antisera directed towards cell-specific markers such as gastrin (G cells), serotonin (EC cells), histidine decarboxylsae (ECL cells) and somatostatin (D cells) to characterize the expression of CgA and CgA-derived peptides in the various endocrine cell populations of the rat stomach. In the oxyntic mucosa, antisera raised against CgA(291-319) and CGA(316-321) immunostained D cells exclusively, whereas antisera raised against bCgA(82-91) and CgA(121-128) immunostained A-like cells and D cells. Antisera raised against CgA(318-349) and CgA(437-448) immunostained ECL cells and A-like cells, but not D cells. In the antrum, antisera against CgA(291-319) immunostained D cells, and antisera against CgA(351-356) immunostained G cells. Our observations suggest that each individual endocrine cell type in the rat stomach generates a unique mixture of CgA-derived peptides, probably reflecting cell-specific differences in the post-translational processing of CgA and its peptide products. A panel of antisera that recognize specific domains of CgA may help to identify individual endocrine cell populations.

Selection of an analytical method for evaluating bovine serum albumin concentrations in pharmaceutical polymeric formulations

Bovine serum albumin (BSA) is a commonly used model protein in the development of pharmaceutical formulations. In order to assay its release from various dosage forms, either the bicinchoninic acid (BCA) assay or a more specific size-exclusion high performance liquid chromatography (SE-HPLC) method are commonly employed. However, these can give erroneous results in the presence of some commonly-used pharmaceutical excipients. We therefore investigated the ability of these methods to accurately determine BSA concentrations in pharmaceutical formulations that also contained various polymers and compared them with a new and compared with a new reverse-phase (RP)–HPLC technique. We found that the RP-HPLC technique was the most suitable method. It gave a linear response in the range of 0.5 -100 µg/ml with a correlation coefficient of 0.9999, a limit of detection of 0.11 µg/ml and quantification of 0.33 µg/ml. The performed ‘t’ test for the estimated and theoretical concentration indicated no significant difference between them providing the accuracy. Low % relative standard deviation values (0.8-1.39%) indicate the precision of the method. Furthermore, the method was used to quantify in vitro BSA release from polymeric freeze-dried formulations.

Reduction of severe bovine serum associated matrix effects on carboxymethylated dextran coated biosensor surfaces

Surface plasmon resonance (SPR) based biosensor technology has been widely used in life science research for many applications. While the advantages of speed, ruggedness, versatility, sensitivity and reproducibility are often quoted, many researchers have experienced severe problem of non-specific binding (NSB) to chip surfaces when performing analysis of biological samples Such as bovine serum. Using the direct measurement of the bovine protein leptin, present in bovine serum samples as a model, a unique buffering system has been developed and optimised which was able to significantly reduce the non-specific interactions of bovine serum components with the carboxymethyl dextran chip (CM5) surface on a Biacore SPR The developed NSB buffering system comprised of HBS-EP buffer, containing 0.5 M NaCl, 0.005% CM-dextran pH 9.0. An average NSB reduction (n = 20) of 85.9% and 87.3% was found on an unmodified CM5 surface and a CM5 with bovine leptin immobilised on the chip surface, respectively. A reduction in NSB of up to 94% was observed on both surfaces. The concentration of the constitutive components and pH of the buffer were crucial in achieving this outcome. (C) 2008 Elsevier B.V. All rights reserved.

Public Administration, Science and Risk Assessment: A Case Study of the U.K. Bovine Spongiform Encephalopathy Crisis

The bovine spongiform encephalopathy (BSE) outbreak in the United Kingdom is regarded as one of the worst public policy crises the British government has experienced during the postwar era. In material terms, it has led to the slaughter of 3.3 million cattle and estimated economic losses of £3.7 billion. In administrative terms, the crisis brought about the dissolution of the Ministry of Agriculture, Fisheries and Food. This article examines the istitutional context in which decisions about the scientific evidence on BSE were made. The authors argue that a centralized system in which government agencies control science for government is inherently vulnerable to alliances of experts and interest groups that undermine the credible assessment of public health and safety risks. Specific societal conditions may encourage risk-opportunistic behavior among policy makers that is conducive to delays and inaction until such time as the evidence of a health risk becomes overwhelming.

The Gastrointestinal Peptide Obestatin Induces Vascular Relaxation Via Specific Activation of Endothelium-Dependent Nitric Oxide Signalling.

Background and purpose: Obestatin is a recently-discovered gastrointestinal peptide with established metabolic actions, which is linked to diabetes and may exert cardiovascular benefits. Here we aimed to investigate the specific effects of obestatin on vascular relaxation. Experimental approach: Cumulative relaxation responses to obestatin peptides were assessed in isolated rat aorta and mesenteric artery (n=8) in the presence/absence of selective inhibitors. Complementary studies were performed in cultured bovine aortic endothelial cells (BAEC). Key results: Obestatin peptides elicited concentration-dependent relaxation in both aorta and mesenteric artery. Responses to full-length obestatin(1-23) were greater than those to obestatin(1-10) and obestatin(11-23). Obestatin(1-23)-induced relaxation was attenuated by endothelial denudation, L-NAME (NO synthase inhibitor), high extracellular K(+) , GDP-ß-S (G protein inhibitor), MDL-12,330A (adenylate cyclase inhibitor), wortmannin (PI3K inhibitor), KN-93 (CaMKII inhibitor), ODQ (guanylate cyclase inhibitor) and iberiotoxin (BK(Ca) blocker), suggesting that it is mediated by an endothelium-dependent NO signalling cascade involving an adenylate cyclase-linked G protein-coupled receptor, PI3K/Akt, Ca(2+) -dependent eNOS activation, soluble guanylate cyclase and modulation of vascular smooth muscle K(+) . Supporting data from BAEC indicated that nitrite production, intracellular Ca(2+) and Akt phosphorylation were increased after exposure to obestatin(1-23). Relaxations to obestatin(1-23) were unaltered by inhibitors of candidate endothelium-derived hyperpolarising factors (EDHFs) and combined SK(Ca) /IK(Ca) blockade, suggesting that EDHF-mediated pathways were not involved. Conclusions and Implications: Obestatin produces significant vascular relaxation via specific activation of endothelium-dependent NO signalling. These actions may be important in normal regulation of vascular function and are clearly relevant to diabetes, a condition characterised by endothelial dysfunction and cardiovascular complications.

Comparison of two polyclonal antibodies for the detection of 19-nortestosterone in bovine bile by ELISA

19-Nortestosterone (beta-NT) is banned for use as a growth promoter in food animals within the European Union. For regulatory control purposes, urine and bile samples are routinely screened by immunoassay. The aim of the present study was to compare the ability of two immunoassays, using two rabbit polyclonal antibodies raised against two different NT derivatives, to detect NT residues in bovine bile. One antiserum cross-reacted with both alpha-NT and beta-NT (alpha/beta-NT), whereas the other was specific for alpha-NT. Bile samples from 266 slaughtered cattle were deconjugated and analyzed using both antibodies, with all screening positives (>2 ng ml(-1)) confirmed by high resolution gas chromatography mass spectrometry. The alpha/beta-NT and alpha-NT antibody-based ELISAs screened 39 and 44 samples positive, respectively, with NT confirmed in 22 and 39, respectively. The alpha/beta-NT antibody-based ELISA produced a false-negative rate of 44% compared to 0% for the alpha-NT antibody-based ELISA. Supplementary investigations concluded that a matrix effect was a major cause of the marked differences in false-negative rates. This result underlines the necessity to validate immunoassays in the sample matrix.

LIQUID-CHROMATOGRAPHY THERMOSPRAY MASS-SPECTROMETRIC ASSAY FOR TRENBOLONE IN BOVINE BILE AND FECES

A liquid chromatography-thermospray mass spectrometric assay was developed and validated to confirm the presence of illegal residues of the synthetic androgenic growth promoter, trenbolone acetate, in cattle. The assay was specific for 17alpha-trenbolone, the major bovine metabolite of trenbolone acetate. Methods were developed for the determination of 17alpha-trenbolone in both bile and faeces, the most appropriate matrices for the control of trenbolone acetate abuse. The clean-up.procedure developed relied on enzymatic hydrolysis, followed by sequential liquid-liquid and liquid-solid extraction. The extracts were then subjected to immunoaffinity chromatography. 17alpha-Trenbolone was detected by selected ion monitoring at m/z 271 using positive ion thermospray ionisation. The limit of detection was approximately 0.5 ng/g in faeces and 0.5 ng/ml in bile.

DETECTION OF CLENBUTEROL RESIDUES IN BOVINE LIVER, MUSCLE, RETINA AND URINE USING GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

A gas chromatographic/mass spectrometric method is described for the detection of clenbuterol residues in liver, muscle, urine and retina. Tissue samples are first digested using protease and any clenbuterol present is extracted using a simple liquid/liquid extraction procedure. The dried extracts are then derivatized using methylboronic acid and the derivatives are subjected to gas chromatography/mass spectrometry on a magnetic sector instrument. The detection limit of the assay is 0.05 ng g-1 clenbuterol in liver, muscle or urine using a 10 g sample size, and 4 ng g-1 in retina using a 0.5 g sample size. The assay is made very specific by using selected ion monitoring of three ions at a resolution of 3500 and by ion ratio measurements. The precision and reproducibility of the assay are enhanced by the use of a deuterated internal standard, with a typical coefficient of variation of 3%.

The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal:GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide

In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of Gal from UDP-Gal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid acceptor. A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity. The normal phenotype was restored by complementing the mutant with the cloned wbbD gene. To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAcalpha-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl(2). Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a beta-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal beta1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.

Production of polyclonal antibodies directed to recombinant methionyl bovine somatotropin

The administration of recombinant methionyl bovine somatotropin (rMbST) to dairy cows to increase milk yield remains a common practice in many countries including the USA, Brazil, Mexico, South Africa and Korea, whereas it has been forbidden within the European Union (EU) since 1999. A rapid screening immunoanalytical method capable of the unequivocal determination of rMbST in milk would be highly desirable in order to effectively monitor compliance with the EU-wide ban for home-made or imported dairy products. For decades, the production of specific antibodies for this recombinant isoform of bovine somatotropin (bST) has remained elusive, due to the high degree of sequence homology between both counterparts (e.g. methionine for rMbST in substitution of alanine in bST at the N-terminus). In this study, we compared several immunizing strategies for the production of specific polyclonal antibodies (pAbs), based on the use of the full-length recombinant protein, an rMbST N-terminus peptide fragment and a multiple antigen peptide (MAP) which consists of an oligomeric branching lysine core attached to the first two N-terminus amino acids of rMbST, methionine and phenylalanine (MF-MAP). The immunization with KLH-conjugated MF-MAP led to the production of the pAb with the highest rMbST/bST recognition ratio amongst the generated battery of antibodies. The pAb exhibited a specific binding ability to rMbST in a competitive antigen-coated ELISA format, which avidity was further improved after purification by rMbST N-terminus peptide-based affinity chromatography. These results suggest that immunodiscrimination between structurally related proteins can be achieved using immuno-enhanced immunogens such as MAPs. © 2012 Elsevier B.V.

Local and systemic immune responses in mice to intranasal delivery of peptides representing bovine respiratory syncytial virus epitopes encapsulated in poly (dl-lactide-co-glycolide) microparticles

The potential of a microparticulate vaccine delivery system in eliciting a specific mucosal antibody response in the respiratory tract of mice was evaluated. Two vaccine candidate peptides representing epitopes from the G attachment and F fusion antigens from bovine respiratory syncytial virus (BRSV) were encapsulated into poly(dl- lactide co-glycolide) biodegradable microparticles. The encapsulation process did not denature the entrapped peptides as verified by detection of peptide-specific antibodies in mucosal secretions by ELISA using peptide as antigen. Following intranasal immunisation, the encapsulated peptides induced stronger upper and lower respiratory tract specific-IgA responses, respectively, than the soluble peptide forms. Moreover, a strong peptide-specific cell-mediated immune response was measured in splenocytes in vitro from the mice inoculated with the encapsulated peptides compared to their soluble form alone indicating that migration of primed T cells had taken place from the site of mucosal stimulation in the upper respiratory tract to the spleen. These results act as a foundation for vaccine efficacy studies in large animal BRSV challenge models.

Tumour necrosis factor-alpha (TNF-alpha) increases nuclear factor kappaB (NFkappaB) activity in and interleukin-8 (IL-8) release from bovine mammary epithelial cells

Epithelia play important immunological roles at a variety of mucosal sites. We examined NFkappaB activity in control and TNF-alpha treated bovine mammary epithelial monolayers (BME-UV cells). A region of the bovine IL-8 (bIL-8) promoter was sequenced and a putative kappaB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NFkappaB activity. As a surrogate marker of NFkappaB activation, we investigated IL-8 release in two models. Firstly in BME-UV monolayers, IL-8 release in the presence of pro- and anti-inflammatory agents was determined by enzyme-linked immunosorbent assay (ELISA). Secondly, we measured IL-8 secretion from a novel model of intact mucosal sheets of bovine teat sinus. IL-8 release into bathing solutions was assessed following treatment with pro- and anti-inflammatory agents. TNF-alpha enhanced NFkappaB activity in bovine mammary epithelial monolayers. p65 NFkappaB homodimer was identified in both control and TNF-alpha treated cells. Novel sequencing of the bovine IL-8 promoter identified a putative kappaB consensus sequence, which specifically bound TNF-alpha inducible p50/p65 heterodimer. TNF-alpha induced primarily serosal IL-8 release in the cell culture model. Pre-treatment with anti-TNF or dexamethasone inhibited TNF-alpha induced IL-8 release. High dose interleukin-1beta (IL-1beta) induced IL-8 release, however significantly less potently than TNF-alpha. Bovine mammary mucosal tissue released high basal levels of IL-8 which were unaffected by TNF-alpha or IL-1beta but inhibited by both dexamethasone and anti-TNF. These data support a role for TNF-alpha in activation of NFkappaB and release of IL-8 from bovine mammary epithelial cells.

Cathelicidin-like Helminth Defence Molecules (HDMs):Absence of Cytotoxic, Anti-microbial and Anti-protozoan Activities Imply a Specific Adaptation to Immune Modulation

Host defence peptides (HDPs) are expressed throughout the animal and plant kingdoms. They have multifunctional roles in the defence against infectious agents of mammals, possessing both bactericidal and immune-modulatory activities. We have identified a novel family of molecules secreted by helminth parasites (helminth defence molecules; HDMs) that exhibit similar structural and biochemical characteristics to the HDPs. Here, we have analyzed the functional activities of four HDMs derived from Schistosoma mansoni and Fasciola hepatica and compared them to human, mouse, bovine and sheep HDPs. Unlike the mammalian HDPs the helminth-derived HDMs show no antimicrobial activity and are non-cytotoxic to mammalian cells (macrophages and red blood cells). However, both the mammalian- and helminth-derived peptides suppress the activation of macrophages by microbial stimuli and alter the response of B cells to cytokine stimulation. Therefore, we hypothesise that HDMs represent a novel family of HDPs that evolved to regulate the immune responses of their mammalian hosts by retaining potent immune modulatory properties without causing deleterious cytotoxic effects.