Kosaku Kobayashi

Purification of canine alpha-fetoprotein and alpha- fetoprotein values in dogs

Canine alpha-fetoprotein (AFP) was purified by a two step method. Anti-dog AFP antiserum was produced by immunizing rabbits with canine fetal serum proteins that failed to bind to an anti-dog whole adult serum affinity column. Canine AFP was then purified from amniotic fluid using affinity chromatography with anti-dog AFP antiserum. The bound protein was then eluted and further purified by passage through an anti-dog whole adult serum column. The non-binding proteins purity and specificity was confirmed by immunoelectrophoresis, double-diffusion, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and cross-reactivity with anti-human AFP. The molecular weight of canine AFP was approximately 66,000 by SDS-PAGE. Normal adult dogs had serum AFP levels of 7-63 ng ml-1. Levels of AFP were not altered by pregnancy but did show a small peak 2 days following parturition. Newborn puppies had serum AFP levels of 14.08 +/- 5.94 mg ml-1 at birth. By 1 week of age, serum AFP had fallen to 0.766 +/- 0.758 mg ml-1. AFP values in newborn puppies are thus considerably higher than those previously reported in humans, pigs and cattle.

Alpha-fetoprotein in serum and tumor tissues in dogs with hepatocellular carcinoma

Serum alpha-fetoprotein (AFP) concentrations were measured before and after surgical removal of tumor masses in four dogs with hepatocellular carcinoma (HCC). Localization of AFP was also examined immunohistochemically in tumor tissues. In three cases, the serum AFP concentration was 10 to 20 times higher than that of normal dogs. One to two months after surgery, the serum AFP concentration had decreased to normal range. AFP was localized in the tumor tissues in these three cases. One case, which had a low serum AFP, did not show AFP localization in tumor tissue.

Development and characterization of monoclonal antibodies against canine trypsin

Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28kDa by SDS-PAGE. Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays.

Development of a canine trypsin-like immunoreactivity assay system using monoclonal antibodies

The radioimmunoassay (RIA) for trypsin-like immunoreactivity (TLI) is one of the most sensitive and specific tests for detecting exocrine pancreatic insufficiency (EPI). An abnormally low serum TLI concentration (<2.5 ng/ml) indicates end-stage EPI. Although RIA methods can be used to detect canine serum TLI, these procedures are beyond the capabilities of most veterinary clinics and general laboratories. Using monoclonal antibodies (mAbs), we developed an enzyme-linked immunosorbent assay (ELISA) for canine TLI and incorporated it into an immunochromatographic test (ICT) for the diagnosis of EPI. The ELISA was linear over TLI concentrations of 1-100 ng/ml. Levels of intra-assay coefficients of variance (CVs) were 1.8-6.1%, inter-assay CVs were 5.1-9.8%, and the recovery of TLI added to two samples of canine serum ranged from 89 to 111 and 93 to 108%, respectively. Good correlation (correlation coefficient, 0.974) occurred between the TLI values obtained by the ELISA method and those by RIA from 56 clinical samples. Serum TLI values in clinically healthy dogs ranged from 7.8 to 29.2 ng/ml by ELISA, and those from dogs with EPI were 0.0-0.6 ng/ml. The values were 0.0-287.4 ng/ml for dogs with pancreatitis, and those from dogs with gastrointestinal disease were 5.5-58.9 ng/ml. The only statistically significant difference (P<0.01) occurred between the TLI level of healthy dogs and those with EPI. The ICT kit showed high reproducibility, and the TLI values yielding negative results differed significantly (P<0.01) from those returning positive results. The ICT kit yielded negative results (indicating EPI) from clinical serum samples with TLI concentrations of 0.0-4.1 ng/ml by ELISA. Both the ELISA and ICT kit are useful tools in the diagnosis of canine EPI.

Relationship between Serum Protein and Loss of Productivity in Dairy Bull Calves for Fattening

Three groups of male Holstein calves were fed for 11 or 12 weeks on different livestock farms . Each group was purchased from a different livestock market and consisted of 10 calves . Body weight, clinical signs and serum protein were periodically evaluated. The electrophoretical patterns and immunoglobulin concentration of serum differed significantly between each group . Two calves of a group that were severely depleted of immunoglobulins died from diarrhoea just after introduction. The survivors in this group were delayed in body weight gain. The proper feeding of colostrum is indispensable to reducing mortality and morbidity following the introduction of dairy bull calves for fattening.