The production and characterisation of dinitrocarbanilide antibodies raised using antigen mimics.

Polyclonal antibodies were produced to detect the coccidiostat nicarbazin. Due to structural constraints of the active component of nicarbazin, dinitrocarbanilide (DNC), three different compounds that shared a common substructure with DNC were used as antigen mimics. The compounds (N-suceinyl-L-alanyl-L-alanyl-L-alanine 4-nitroanilide (SAN), L-glutamic acid gamma-(p-nitroanilide) (GAN) and p-nitrosuccinanilic acid (NSA)) were conjugated to a carrier protein and used in the immunisation of rabbits. Five different polyclonal sera were produced and consequently characterised. The antibodies exhibited an IC50 range of 2.3-7.6 ng/ml using a competitive ELISA procedure, Serum from one rabbit, R555, exhibited an IC50 of 2.9 ng/ml for DNC and cross-reactivity studies showed that this serum was specific for DNC and did not cross-react with other coccidiostats such as halofuginone, toltrazuril or ronidazole. (C) 2002 Elsevier Science B.V. All rights reserved.

The production and characterisation of an antibody to detect the coccidiostat toltrazuril and its metabolite ponazuril.

The production of an antibody to detect toltrazuril or its metabolite ponazuril is complicated due to structural constraints of conjugating these coccidiostats to a carrier protein. Therefore a search was carried out for a compound that shared a common substructure to use as an antigen mimic. The chosen compound, trifluoraminoether, was conjugated to two carrier proteins (HSA and BTG) and used in the immunisation of six rabbits. Two immunogen doses (1 mg and 0.1 mg) were also used. All six rabbits produced an immunological response to the hapten regardless of the carrier protein or immunogen dose used. The most sensitive polyclonal antibody produced, designated R609, was subsequently characterised. This antiserum exhibited an IC50 of 18 ng ml-1 using a competitive ELISA format. Cross reactivity studies show that this serum is specific for toltrazuril and its metabolites (toltrazuril sulfoxide and toltrazuril sulfone) but does not cross-react with other coccidiostats such as halofuginone, nitroimidazoles or nicarbazin. This is the first reported production of an antibody capable of specifically binding toltrazuril and ponazuril.

Production and characterisation of polyclonal antibodies to a range of nitroimidazoles.

The nitroimidazoles dimetridazole and ronidazole are metabolised to hydroxydimetridazole, while metronidazole is metabolised to hydroxymetronidazole. To screen for a large number of samples by immunoassay for the presence of this family of drugs and metabolites, it was necessary to produce an antibody with broad-spectrum recognition. Metronidazole and hydroxydimetridazole were selected as antigens as they could be coupled to large (immunogenic) carrier proteins at two different positions of the general nitroimidazole structure. The resulting conjugates were used to immunise rabbits, sheep and goats. Seventeen out of thirty-nine animals immunised produced a detectable antibody titre and these antibodies were consequently characterised as regards sensitivity and cross-reactivity.

The panel of antisera produced exhibited IC50 ranging from 1.26 to 73.76 ng ml-1 using a competitive ELISA assay. Cross-reactivity studies showed that sera from several animals were capable of significant binding of six of the seven nitroimidazole compounds tested.

Heterogeneity in the granulomatous response to mycobacterial infection in patients with defined genetic mutations in the interleukin 12-dependent interferon-gamma production pathway

Granulomatous Diseases Review Heterogeneity in the granulomatous response to mycobacterial infection in patients with defined genetic mutations in the interleukin 12-dependent interferon-gamma production pathway

New Zealand chironomids as proxies for human-induced and natural environmental change: Transfer functionsfor temperature and lake production (chlorophyll a)

The analysis of chironomid taxa and environmental datasets from 46 New Zealand lakes identified temperature (February mean air temperature) and lake production (chlorophyll a (Chl a)) as the main drivers of chironomid distribution. Temperature was the strongest driver of chironomid distribution and consequently produced the most robust inference models. We present two possible temperature transfer functions from this dataset. The most robust model (weighted averaging-partial least squares (WA-PLS), n = 36) was based on a dataset with the most productive (Chl a > 10 lg l)1) lakes removed. This model produced a coefficient of determination (r2 jack) of 0.77, and a root mean squared error of prediction (RMSEPjack) of 1.31C. The Chl a transfer function (partial least squares (PLS), n = 37) was far less reliable, with an r2 jack of 0.49 and an RMSEPjack of 0.46 Log10lg l)1. Both of these transfer functions could be improved by a revision of the taxonomy for the New Zealand chironomid taxa, particularly the genus Chironomus. The Chironomus morphotype was common in high altitude, cool, oligotrophic lakes and lowland, warm, eutrophic lakes. This could reflect the widespread distribution of one eurythermic species, or the collective distribution of a number of different Chironomus species with more limited tolerances. The Chl a transfer function could also be improved by inputting mean Chl a values into the inference model rather than the spot measurements that were available for this study.

Degradation, cyclic adenosine monophosphate production, insulin secretion, and glycemic effects of two novel N-terminal Ala(2)-substituted analogs of glucose-dependent insulinotropic polypeptide with preserved biological activity in vivo

Glucose-dependent insulinotropic polypeptide (GIP) has significant potential in diabetes therapy due to its ability to serve as a glucose-dependent activator of insulin secretion. However, its biological activity is severely compromised by the ubiquitous enzyme dipeptidylpeptidase IV (DPP IV), which removes the N-terminal Tyr(1)-Ala(2) dipeptide from GIP. Therefore, 2 novel N-terminal Ala(2)-substituted analogs of GIP, with Ala substituted by 2-aminobutyric acid (Abu) or sarcosine (Sar), were synthesized and tested for metabolic stability and biological activity both in vitro and in vivo. Incubation with DPP IV gave half-lives for degradation of native GIP, (Abu(2))GIP, and (Sar(2))GIP to be 2.3, 1.9, and 1.6 hours, respectively, while in human plasma, the half-lives were 6.2, 7.6, and 5.4 hours, respectively. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, native GIP, (Abu(2))GIP, and (Sar(2))GIP dose-dependently stimulated cyclic adenosine monophosphate (camp) production with EC50 values of 18.2, 38.5, and 54.6 nmol/L, respectively. In BRIN-BD11 cells, both (Abu(2))GIP and (Sar(2))GIP (10(-13) to 10(-8) mol/L) dose-dependently stimulated insulin secretion with significantly enhanced effects at 16.7 mmol/L compared with 5.6 mmol/L glucose. In obese diabetic (ob/ob) mice, GIP and (Sar(2))GIP significantly increased (1.4-fold to 1.5-fold; P <.05) plasma insulin concentrations, whereas (Abu(2))GIP exerted only minor effects. Changes in plasma glucose were small reflecting the severe insulin resistance of this mutant. The present data show that substitution of the penultimate N-terminal Ala(2) in GIP by Abu or Sar results in analogs with moderately reduced metabolic stability and biological activity in vitro, but with preserved biological activity in vivo. (C) 2003 Elsevier Inc. All rights reserved.

Characterisation of the Interaction of Lactate Dehydrogenase With Tween-20 Using Isothermal Titration Calorimetry, Interfacial Rheometry and Surface Tension Measurements

In this study the nature of the interaction between Tween-20 and lactate dehydrogenase (LDH) was investigated using isothermal titration calorimetry (ITC). In addition the effects of the protein and surfactant on the interfacial properties were followed with interfacial rheology and surface tension measurements in order to understand the mechanism by which the surfactant prevents protein adsorption to the air– water interface. Comparisons were made with Tween-40 and Tween-80 in order to further investigate the mechanism. ITC measurements indicated a weak, probably hydrophobic, interaction between Tween-20 and LDH. Prevention of LDH adsorption to the air–water interface by the Tween surfactants was correlated with surface energy rather than surfactant CMC. While surface pressure appears to be the main driving force for the displacement of LDH from the air–water interface by Tween-20 a solubilisation mechanism may exist for other protein molecules. More generally the results of this study highlight the value of the use of ITC and interfacial measurements in characterising the surface behaviour of mixed surfactant and protein systems.