Immobilization of an esterase inhibitor on a porous hollow-fiber membrane by radiation-induced graft polymerization for developing a diagnostic tool for feline kidney diseases.

Removal of the major urinary protein, cauxin, a carboxylesterase, from cat urine is essential for distinguishing between physiological and abnormal proteinuria by a urine dipstick. We have previously developed a material for removing cauxin by using lens culinaris agglutinin (LCA) lectin which targets the N-linked oligosaccharides present in cauxin. To improve the affinity and specificity toward cauxin, we immobilized 1,1,1-trifluoro-3-(2-sulfanylethylsulfanyl) propane-2-one, an inhibitor of esterases, to a polymer chain grafted on to a porous hollow-fiber membrane by applying radiation-induced graft polymerization. Normal male urine was forced to permeate through the pores rimmed by the ligand-immobilized polymer chain. Cauxin could not be detected in the effluent from the membrane. The residence time of the urine across a membrane thickness of 1 mm was set at 7 s. The respective dynamic and equilibrium binding capacities of the membrane for cauxin were 2 and 3 mg/g. The developed cauxin-affinity membrane material was more effective for diagnosing cat kidney diseases than the LCA lectin tip.

Establishment of a quantitative ELISA for the measurement of allergen-specific IgE in dogs using anti-IgE antibody cross-reactive to mouse and dog IgE.

As IgE plays a pivotal role in type I hypersensitivity-mediated allergic diseases, it is valuable to measure absolute quantity of serum antigen-specific IgE for clinical and research purposes. Here we describe a novel ELISA system that enables quantification of antigen-specific IgE in ng/ml in dogs. A newly developed monoclonal antibody (CRE-DM) was shown to recognize canine and mouse IgE equally in a dose dependent manner, but it did not recognize canine IgG. The reactivity of CRE-DM to canine IgE was also confirmed by an inhibition ELISA using canine IgE as an inhibitor and the maximum inhibition rate was 91.3%. In order to know whether canine IgE specific to an allergen could be quantitatively measured with an ELISA using CRE-DM, we established a quantitative ELISA that could measure canine IgE recognizing Cry j 1, one of the major allergens of Japanese cedar pollen. In this ELISA, a standard curve was created by using concentration-predetermined Cry j 1-specific monoclonal mouse IgE. According to the standard curve, the concentration of Cry j 1-specific IgE in dogs that were experimentally sensitized to Japanese cedar pollen could be calculated and determined in ng/ml. The specificity of the Cry j 1-specific IgE ELISA using CRE-DM was also confirmed by inhibition ELISA using canine IgE as an inhibitor and the inhibition rate was 97.0%. Reproducibility of the ELISA in three independent assays was determined using groups of pooled canine sera whose Cry j 1-IgE titers ranged from 155.9 to 888.2 ng/ml. Intra- and inter-assay reproducibility was determined with coefficient of variation ranging between 3.1-5.2% and 2.2-8.0%, respectively. These results demonstrated that the ELISA utilizing CRE-DM was a specific, reliable and robust new laboratory test that could quantify absolute amount of antigen-specific IgE in canine serum. The ELISA will serve as a useful tool in the clinics to evaluate the change of serum IgE titers during anti-allergic treatments as well as during seasonal fluctuation of allergen exposure.

Screening for proteinuria in cats using a conventional dipstick test after removal of cauxin from urine with a Lens culinaris agglutinin lectin tip.

Proteinuria is an important indicator of urinary tract disease and urine dipsticks are simple and sensitive tools to screen for this marker. However, the use of dipsticks to screen for proteinuria may not be appropriate in cats, since cauxin, a 70 kDa glycoprotein, is secreted by the kidneys in clinically normal animals of this species. To circumvent this problem, a Lens culinaris agglutinin (LCA) lectin tip was developed to remove cauxin from feline urine, followed by conventional urine dipstick testing for proteinuria. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue R-250 staining indicated that >90% cauxin in the urine of 13 clinically normal cats was trapped by the LCA lectin tip, so that the dipstick protein 'score' changed from 'positive' (≥30 mg/dL) for untreated urine to 'negative' (≤10 mg/dL) for lectin tip-treated urine. In contrast, SDS-PAGE indicated that lectin tip-treated samples from 20 animals with renal disease contained high concentrations of albumin and low-molecular weight proteins; dipstick testing of lectin tip-treated urine resulted in a consistently positive protein score. The accuracy of the dipstick method for detecting cats with abnormal proteinuria is enhanced if dipsticks are used with urine samples that have first been passed through the LCA lectin tip.

Isolation and expression of five-amino-acid-deleted variant of feline hepatocyte growth factor (HGF) cDNA.

Hepatocyte growth factor (HGF) is a pleiotropic cytokine that stimulates a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial and hematopoietic cells. We have cloned a different form of cDNA, with a deletion of 15 base pairs predicted to result in the loss of 5 amino acids from the first kringle domain. To investigate the biological activity, original and deleted variant of feline HGF cDNAs were transiently expressed in COS-7 cells. Both recombinant feline HGFs showed almost the same dose-response curves in the stimulation of the growth of BNL CL.2 cells (a mouse hepatocyte cell line) and scatter activity of Madin-Darby canine kidney (MDCK) cells. The findings reported here suggest that the deleted variant of feline HGF has almost the same biological activity as the original in terms of the proliferation and scatter activity.

Canine hepatocyte growth factor: molecular cloning and characterization of the recombinant protein.

Hepatocyte growth factor (HGF) is a pleiotropic cytokine originally identified and cloned as a potent mitogen for hepatocytes. The HGF receptor is the transmembrane tyrosine kinase encoded by c-MET proto-oncogene. Various lines of evidence suggest that the HGF/c-MET receptor system plays essential roles in monocyte-macrophage function, mammalian development, angiogenesis and organ regeneration. We have cloned canine HGF (CaHGF) cDNA from leukocytes by the methods of reverse transcription (RT)-polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). Canine HGF contains an open reading frame (ORF) of 2193 nucleotides, coding for 730 amino acids. The deduced amino acid sequence of canine HGF shows 97.5, 92.3, 92.1, and 92.0% homologies with those of feline, human, mouse, and rat, respectively. The possible glycosylation sites, cysteine residues linking the alpha and beta chains and the proteolytic processing site are conserved in all species. In addition, we have found a variant cDNA that deleted a sequence of 15 base pairs in the first kringle domain (K1) and resulted in the deletion of five amino acids. To confirm the biological activities of canine HGF cDNAs, both cDNAs were transiently expressed in COS-7 cells. The conditioned medium from the canine HGF-transfected COS-7 cells stimulated the growth of BNL CL.2 cells (a mouse hepatocyte cell) and scattering activity of Madin-Darby canine kidney (MDCK) cells. The materials reported here will be a crucial resource for further studies of canine HGF.