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 > or = 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 125I. 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.
Publication Date: 2003-06-07 PubMed ID: 12791241DOI: 10.1051/vetres:2003007Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The researchers have successfully purified equine trypsin and developed a technique to measure its presence in body fluids, particularly in situations of shock where high levels of trypsin indicate pancreatic damage. This technique could help in understanding and diagnosing conditions like equine intestinal obstruction.
Purification and Development of Radioimmunoassay
- The process began with the purification of trypsin from the pancreatic tissue of a horse. This was achieved through multiple steps including extraction in an acid medium, precipitation with ammonium sulfate, and chromatographic methods.
- The purified trypsin then underwent gel polyacrylamide electrophoresis and was identified as a monomeric enzyme with a molecular weight of 27 kDa.
- In order to create a radioimmunoassay (RIA) technique for measuring equine trypsin-like immunoreactivity (TLI), the isolating trypsin was used to immunize rabbits, creating a specific antiserum (an antibody-rich solution).
- The trypsin was also labelled with iodine-125, a radioactive isotope, to create the antigen which the RIA can detect.
Implementation of Radioimmunoassay
- The RIA technique involves the binding of the trypsin antigen to the antiserum antibody, forming an antigen-antibody complex.
- This complex is separated from the mixture using sheep anti-rabbit gammaglobulins, and the remaining radioactivity is measured to determine the presence and concentration of trypsin.
- The researchers found the RIA technique to have good sensitivity, accuracy, and precision, as well as reproducibility.
Pancreas Role in Equine Intestinal Obstruction
- Using this RIA technique, high levels of trypsin were detected in the plasma of horses with strangulating intestinal obstruction, suggesting a potential role of pancreatic damage in this condition.
- The researchers suggest further studies and enzyme assays should be conducted to establish the pancreas’ role in equine intestinal obstruction. They believe that this new method will be a valuable tool in such studies.
Cite This Article
APA
Grulke S, Deby-Dupont G, Gangl M, Franck T, Deby C, Serteyn D.
(2003).
Equine trypsin: purification and development of a radio-immunoassay.
Vet Res, 34(3), 317-330.
https://doi.org/10.1051/vetres:2003007 Publication
Researcher Affiliations
- Anesthésiologie générale et Pathologie chirurgicale des Grands Animaux, B41, Faculté de Médecine Vétérinaire, Université de Liège, 4000 Sart Tilman, Belgium. sgrulke@ulg.ac.be
MeSH Terms
- Animals
- Chromatography, Gel
- Electrophoresis, Polyacrylamide Gel
- Horse Diseases / blood
- Horses
- Immune Sera / immunology
- Intestinal Obstruction / blood
- Intestinal Obstruction / veterinary
- Iodine Radioisotopes
- Radioimmunoassay / methods
- Reference Values
- Reproducibility of Results
- Sensitivity and Specificity
- Trypsin / blood
- Trypsin / isolation & purification
- Trypsin / metabolism
Citations
This article has been cited 1 times.- Bridges CS, Miller PS, Lidbury JA, Suchodolski JS, Yi Y, Engelhardt JF, Steiner JM. Validation of a radioimmunoassay of serum trypsin-like immunoreactivity in ferrets. J Vet Diagn Invest 2018 Jul;30(4):517-522.
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