Biosensor immunoassay of ivermectin in bovine milk

A rapid and sensitive biosensor immunoassay was developed for determination of ivermectin residues in bovine milk. A detection limit of 16.2 ng/mL was achieved. A Biacore optical biosensor based on surface plasmon resonance was used, and a range of extraction techniques was investigated. In the final assay procedure, ivermectin was extracted with acetonitrile followed by C-8 solid-phase extraction cleanup. It was proven experimentally that 2 methods of milk storage, freezing or addition of mercury-containing compounds as preservatives, could be used without considerable change in detected concentrations (samples were fortified with ivermectin after storage). The average values for milk samples spiked at 100 and 50 ng/mL concentrations were 102.6 and 51.5 ng/mL, respectively. Extraction and analysis of 20 milk samples were performed within a single working day.

DETECTION OF IRRADIATED CHICKEN MEAT BY ANALYSIS OF LIPID EXTRACTS FOR 2-SUBSTITUTED CYCLOBUTANONES USING AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY

The means to detect the irradiation of food has been investigated for many years. In recent times radiolytic products, termed 2-alkylcyclobutanones (2-CBs), have been identified as excellent markers of irradiation in lipid-containing foods. An ELISA test was developed, which was capable of detecting a number of these compounds in irradiated chicken meat. A polyclonal antiserum was raised to a 2-CB containing a terminal carboxyl group conjugated to a carrier protein. This antiserum was highly specific for cyclobutanones containing C-10 and C-12 side chains. During assay validation the limit of detection of the assay was calculated to be 0.064 pg of 2-CB per gram of fat, within- and between-assay variations ranged from 6.7 to 18%. During experimental studies, chicken meat irradiated at doses ranging from 2.5 to 10 kGy were assayed and correctly identified as being treated. Quantitative comparisons between the ELISA and CC-MS revealed a good correlation (r(2) = 0.913) between the two methodologies in concentrations of 2-CB detected in irradiated samples.

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.

Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers in cancer progression and survival: a systematic review

Objective: To investigate the association between angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) and disease progression and survival in cancer patients.

Methods: Using terms for cancer and ACEIs/ARBs, MEDLINE, EMBASE and Web of Science were systematically searched for observational/interventional studies that used clinically relevant outcomes for cancer progression and survival.

Results: Ten studies met the inclusion criteria. Two studies showed a significant improvement in overall survival (OS) with ACEI/ARB use among patients with advanced pancreatic (HR 0.52, 95% CI 0.29–0.88) and non-small cell lung cancer (HR 0.56, 95% CI 0.33–0.95). An improvement in progression-free survival (PFS) was also reported for pancreatic cancer patients (HR 0.58, 95% CI 0.34–0.95) and patients with renal cell carcinoma (HR 0.54, p = 0.02). ACEI/ARB use was protective against breast cancer recurrence (HR 0.60, 95% CI 0.37–0.96), colorectal cancer distant metastasis (OR 0.22, 95% CI 0.08–0.65) and prostate specific antigen (PSA) failure in prostate cancer patients (p = 0.034). One study observed a worse OS (HR 2.01, 95% CI 1.00–4.05) and PFS in ACEI users with multiple myeloma (p = 0.085) while another reported an increased risk of breast cancer recurrence (HR = 1.56, 95% CI 1.02–2.39).

Conclusion: There is some evidence to suggest that ACEI or ARB use may be associated with improved outcomes in cancer patients. Larger, more robust studies are required to explore this relationship further.

The purification and properties of phosphonoacetate hydrolase, a novel carbon-phosphorus bond-cleavage enzyme from Pseudomonas fluorescens 23F

A novel, inducible, carbon-phosphorus bond-cleavage enzyme, phosphonoacetate hydrolase, was purified from cells of Pseudomonas fluorescens 23F grown phosphonoacetate. The native enzyme had a molecular mass of approximately 80 kDa and, upon SDS/PAGE, yielded a homogenous protein band with an apparent molecular mass of about 38 kDa. Activity of purified phosphonoacetate hydrolase was Zn2+ dependent and showed pH and temperature optima of approximately 7.8 and 37 degrees C, respectively. The purified enzyme had an apparent K-m of 1.25 mM for its sole substrate phosphonoacetate, and was inhibited by the structural analogues 3-phosphonopropionate and phosphonoformate. The NH2-terminal sequence of the first 19 amino acids displayed no significant similarity to other databank sequences.

Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

Structure and function of sedoheptulose-7-phosphate isomerase, a critical enzyme for lipopolysaccharide biosynthesis and a target for antibiotic adjuvants

The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of L-glycero-D-manno-heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-manno-heptose 7-phosphate, the first committed step in the formation of ADP-heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism.

Functional characterization of the initiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-l-Rhap-(1-->], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three alpha-l-rhamnose residues, and a beta-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.

Outer membrane protein profiles and multilocus enzyme electrophoresis analysis for differentiation of clinical isolates of Proteus mirabilis and Proteus vulgaris

Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and three MP profiles, respectively. The remaining P. mirabilis and P. vulgaris isolates had unique profiles. MEE analysis was used to further discriminate among the strains belonging to the same MP groups. Thirty-five distinct electrophoretic types (ETs) were identified among P. mirabilis isolates. The isolates of P. mirabilis from the four most common MP groups were subgrouped into 30 ETs. All of the P. vulgaris strains had unique ETs. The results suggest that upon biochemical classification of Proteus isolates as P. mirabilis or P. vulgaris, further differentiation among strains of the same species can be obtained by the initial determination of MP profiles followed by MEE analysis of strains with identical MPs.

A PLATE ASSAY FOR THE DETECTION OF ORGANOPHOSPHONATE MINERALIZATION BY ENVIRONMENTAL BACTERIA, AND ITS MODIFICATION AS AN ACTIVITY STAIN FOR IDENTIFICATION OF THE CARBON-PHOSPHORUS BOND-CLEAVAGE ENZYME PHOSPHONOACETATE HYDROLASE

A replica plate screening technique, based on the acid molybdate assay for detection of phosphate has been developed to permit the detection of microorganisms capable of mineralizing organophosphonates. The method was further adapted as the basis of an activity stain for the detection of the carbon - phosphorus bond cleavage enzyme phosphonoacetate hydrolase in PAGE gels.

Detection of Paralytic Shellfish Toxins by a Solid-Phase Inhibition Immunoassay Using a Microsphere-Flow Cytometry System

Paralytic shellfish poisoning is a toxic syndrome described in humans following the ingestion of seafood contaminated with saxitoxin and/or its derivatives. The presence of these toxins in shellfish is considered an important health threat and their levels in seafood destined to human consumption are regulated in many countries, as well as the levels of other chemically unrelated toxins. We studied the feasibility of immunodetection of saxitoxin and its analogs using a solid-phase microsphere assay coupled to flow cytometry detection in a Luminex 200 system. The technique consists of a competition assay where the toxins in solution compete with bead-bound saxitoxin for binding to an antigonyautoxin 2/3 monoclonal antibody (GT-13A). The assay allowed the detection of saxitoxin both in buffer and mussel extracts in the range of 2.2-19.7 ng/mL (IC(20)-IC(80)). Moreover, the assay cross-reactivity with other toxins of the group is similar to previously published immunoassays, with adequate detection of most analogs except N-1 hydroxy analogs. The recovery rate of the assay for saxitoxin was close to 100%. This microsphere-based immunoassay is suitable to be used as a screening method, detecting saxitoxin from 260 to 2360 µg/kg. This microsphere/flow cytometry system provided similar sensitivities to previously published immunoassays and provides a solid background for the development of easy, flexible multiplexing of toxin detection in one sample.

Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs

Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microspheres for the detection of six selected priority coccidiostats. Different coccidiostats, with and without carrier proteins, were covalently coupled onto different bead sets and tested in combination with polyclonal antisera and with a fluorescent-labelled secondary antibody. The five optimal combinations were selected for this multiplex and a simple-to-use sample extraction method was applied for screening blank and spiked eggs and feed samples. A very good correlation (r ranging from 0.995 to 0.999) was obtained with the responses obtained in two different flow cytometers (Luminex 100 and FLEXMAP 3D). The sensitivities obtained were in accordance with the levels set by the EU as the measured limits of detection for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (4,4'-dinitrocarbanilide) and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 µg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 µg/kg, respectively.

Genetic variation in MME in relation to neprilysin protein and enzyme activity, Aβ levels, and Alzheimer's disease risk

Neprilysin (NEP), also known as membrane metalloendopeptidase (MME), is considered amongst the most important ß-amyloid (Aß)-degrading enzymes with regard to prevention of Alzheimer's disease (AD) pathology. Variation in the NEP gene (MME) has been suggested as a risk factor for AD. We conducted a genetic association study of 7MME SNPs - rs1836914, rs989692, rs9827586, rs6797911, rs61760379, rs3736187, rs701109 - with respect to AD risk in a cohort of 1057 probable and confirmed AD cases and 424 age-matched non-demented controls from the United Kingdom, Italy and Sweden. We also examined the association of these MME SNPs with NEP protein level and enzyme activity, and on biochemical measures of Aß accumulation in frontal cortex - levels of total soluble Aß, oligomeric Aß(1-42), and guanidine-extractable (insoluble) Aß - in a sub-group of AD and control cases with post-mortem brain tissue. On multivariate logistic regression analysis one of the MME variants (rs6797911) was associated with AD risk (P = 0.00052, Odds Ratio (O.R. = 1.40, 95% confidence interval (1.16-1.70)). None of the SNPs had any association with Aß levels; however, rs9827586 was significantly associated with NEP protein level (p=0.014) and enzyme activity (p=0.006). Association was also found between rs701109 and NEP protein level (p=0.026) and a marginally non-significant association was found for rs989692 (p=0.055). These data suggest that MME variation may be associated with AD risk but we have not found evidence that this is mediated through modification of NEP protein level or activity.