Equine trypsin: purification and development of a radio-immunoassay

Abstract

-Shock is accompanied by generalised splanchnic hypoperfusion, and splanchnic organs like the pancreas can be damaged, as shown in animal experimental models and in humans, by the presence of high plasma concentrations of trypsin and other pancreatic enzymes. In order to design a radioimmunoassay technique (RIA) for the measurement of equine trypsin-like immunoreactivity (TLI) in biological fluids, trypsin was purified (with purity 96 %) from the equine pancreas by extraction in an acid medium, ammonium sulfate precipitations, gel filtration chromatography and, after activation of trypsinogen into trypsin, affinity chromatography. Gel polyacrylamide electrophoresis showed a monomeric enzyme with a molecular weight of 27 kDa. The purified equine trypsin served for the immunisation of rabbits in order to obtain a specific antiserum, and the labelled antigen was prepared by iodination of equine trypsin with 125 I. The RIA was based on the binding of the antigen to the antibody followed by the separation of the antigen-antibody complex by immunoprecipitation in the presence of sheep anti-rabbit gammaglobulins and the assay of the radioactivity in the precipitate. The RIA showed good sensitivity, specificity, precision, accuracy and reproducibility. The reference mean value of TLI in the plasma of healthy horses (n = 20) was 30.01 ± 6.84 ng/mL (upper confidence limit 50.52 ng/mL; p < 0.01). Three horses with non strangulating intestinal obstruction without shock showed TLI values within normal limits whereas 5 of 7 horses with strangulation obstruction showed TLI levels above the upper confidence limit. Further studies using the RIA and the enzymatic assay should be performed in order to confirm the role of the pancreas in equine intestinal obstruction. equine trypsin / purification / radio-immunoassay / reference range / intestinal obstruction ³

EquiNox2: A new method to measure NADPH oxidase activity and to study effect of inhibitors and their interactions with the enzyme

“…Among the enzymes producing ROS, implied in human [1] or horse pathologies [10], NADPH oxidase is an important therapeutic target as it produces O 2 •-from which derives most of the other ROS [4]. To study the effect of potential inhibitors on NADPH oxidase activity, there are many indirect methods based on the measurement of O 2 •-produced by whole cells, on the in vitro reconstitution of the enzyme using cell-free systems [12,13,24] or on the determination of the expression rate of the enzyme [25].…”

“…In some pathological conditions of acute or chronic inflammation, the uncontrolled activation of PMNs is responsible for ROS releases in the extracellular medium causing deleterious effects on neighbouring cells. Unlike other species, horses are particularly sensitive to inflammation [8] and suffer from inflammatory pathologies such as laminitis, recurrent airway obstruction or intestinal strangulation that are often associated with an endotoxic shock [9,10]. Several research groups highlighted the PMNs implication in these pathologies, with a release of inflammatory mediators, myeloperoxidase, proteases, ROS,… It appears that the modulation of enzymes involved in neutrophil stimulation and ROS production such as Nox2 becomes therapeutic targets to treat pathologies with excessive inflammation [4,5,11].…”

Validation of a radioimmunoassay of serum trypsin-like immunoreactivity in ferrets

“…To date, there are three other species besides humans for which an immunoassay for the measurement of TLI in serum has been developed: dogs, cats, and horses. 6,17,22 Despite ferrets being a well-known animal model for human CF and a potential animal model for human pancreatitis, to the authors’ knowledge, there is no immunoassay available for the measurement of serum TLI concentrations in ferret ( Mustela putorius furo ) serum. 20 Therefore, the objective of the current study was to develop and analytically validate a radioimmunoassay (RIA) for the measurement of TLI in serum from ferrets.…”