• Journal of Veterinary Clinics
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• v.25 no.5
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• pp.317-323
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• 2008

Canine trypsin-like immunoreactivity (cTLI), which is a mirror of the concentration of trypsin and trypsinogen, is a pancreas-specific enzyme and a suitable marker for canine pancreatitis and especially exocrine pancreatic insufficiency (EPI). To develop the immunochromatographic test kit, monoclonal antibodies that recognize cTLI were prepared. Anionic trypsin, cationic trypsin, and chymotrypsin from canine pancreas were successfully purified to homogeneity, using ammonium sulfate fractionation and benzamidine-affinity chromatography. The purification fold for anionic trypsin was 108 times when compared with that of the homogenation of pancreas. The molecular weights by SDS-PAGE analysis were approximately 23 kDa for chymotrypsin and approximately 20 kDa for cationic trypsin and anionic trypsin, respectively. Using the purified trypsin-like proteins, ten hybridomas which secret canine trypsin-specific monoclonal antibody were prepared. Klotz plot indicated that hybridomas, 5G2H10G4 and 2F4A11, have high affinity constant (Ka) of $4.1\;{\times}\;10^{9}$ and $1.8\;{\times}\;10^{9}$, respectively. Especially, 5F9H3 showed the cationic typsin-specific binding pattern and its Ka was determined to $4.5\;{\times}\;10^{9}$. The development of immunochromatographic test kit using these monoclonal antibodies against cTLI will be very useful in the diagnosis of canine EPI or canine pancreatitis.

• Proceedings of the Korean Society of Veterinary Clinics Conference
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• 2008.05a
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• pp.124-124
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• 2008

• KSBB Journal
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• v.5 no.3
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• pp.215-221
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• 1990

The solubilization of BSA, pepsin, trypsin and ribonuclease-a in cationic reverse micellar solutions has been investigated. For complete solubilization into reverse micellar solutions, pH values of several pH units above the isoelectric point of each protein were required. Solubilization was observed to decrease with increasing ionic strength of KCI. The size of empty micelles showed no significant change with increasing ionic strength. Trioctylmethyl ammonium chloride (TOMAC) in cyolohexane showed the lowest solubilization for the proteins. The system didodecyl dimethyl ammonium bromide (DDAB)-tetrachloroethylene was capable of solubilizing larger amounts of proteins than the system DDAB-benzene. Cetyl trimethyl ammonium bromide(CTAB)-hexanol-isooctane system showed lower solubilization than DDAB-tetrachloroethylene system, while it had higher Wo value.