Immunochemical study of canine intestinal, hepatic, and osseous alkaline phosphatases.

Partially purified alkaline phosphatase (ALP) from canine intestine, liver, and bone were injected into rabbits to elicit anti-canine intestinal, hepatic, and osseous ALP antibodies, respectively. The antibody formed a soluble enzyme-antienzyme complex when directly interacted with the ALP antigen. In order to form an insoluble complex, it was then necessary to interact the initial soluble complex with the goat anti-rabbit gamma-globulin antibody in the 2nd step. Antiintestinal ALP antibody was highly specific and did not cross react with canine hepatic, osseous, splenic, and renal ALP. Antiliver and antibone ALP antibodies, on the other hand, did cross react with hepatic, osseous, splenic, and renal ALP, but not with the intestinal ALP.     

Canine serum thermostable alkaline phosphatase isoenzyme from a dog with hepatocellular carcinoma

A dog histopathologically diagnosed with hepatocellular carcinoma (HCC) showed very high serum alkaline phosphatase (ALP) activity. A supernatant of ascitic fluid and tumor tissue extracted from the dog also showed much higher ALP activity than normal. ALP isoenzyme analysis of samples was performed using polyacrylamide gel disk electrophoresis, and a wide, broad abnormal band was observed. By various treatments, the abnormal band showed thermostability, which is a characteristic of tumor-associated ALP that has only been reported in humans. The thermostable ALP isoenzyme was not found in sera from 39 dogs with several types of tumor that originated from the liver, except for HCC, nor was it found in 10 dogs with hepatic diseases that did not include hepatic tumors. The thermostable ALP isoenzyme seemed to be associated with canine HCC.     

Alkaline phosphatase isoenzymes in feline serum using an agarose gel alkaline phosphatase kit method.

Total serum alkaline phosphatase (ALP) activity is the product of the combined activity of isoenzymes from a number of tissue sources. In this study, a commercially available kit for electrophoretic separation of ALP isoenzymes in an agarose gel was used to separate ALP isoenzymes in feline tissue extracts and serum. Five separate bands of ALP activity were identified. These bands were numbered 1 to 5 with band 1 having the most anodal migration. The tissue of origin corresponding to the migration position of the isoenzymes are as follows: Band 3 was the liver isoenzyme, band 4 was the bone isoenzyme and ALP isoenzymes of both intestine and kidney migrate in the position labelled band 5. Band 1 appears to be related to albumin and does not represent true ALP activity. The tissue source of band 2 (a and b) was not identified. Serum ALP activity of mature, healthy cats is primarily of liver origin. Immature cats (< 1 year of age) have a greater proportion of the bone isoenzyme in the serum.     

Semiautomated analysis of alkaline phosphatase isoenzymes in serum of normal cynomolgus monkeys (Macaca fascicularis)

Alkaline phosphatase (ALP) isoenzyme analysis, using a combination of wheat germ lectin (WGL) precipitation, levamisole inhibition and an automated p-nitrophenylphosphate assay was validated for use with serum from monkeys (Macaca fascicularis) and used to determine the activities of liver ALP (LALP), bone ALP (BALP) and intestinal ALP (IALP). Based on serial dilution studies and within-run and between-run coefficients of variation, each assay had excellent linearity and acceptable precision. In addition, liver and intestinal mucosa extracts for tissue specific alkaline phosphatases were used to confirm assay validations. Gender-specific differences for total ALP, LALP, and BALP activities were present in sera from normal monkeys between 2 and 4 years of age. Males had 1.3-fold higher total ALP and LALP activities, and 1.5-fold higher BALP activity compared with females. The majority of ALP activity in normal monkey serum was LALP isoenzyme activity, which ranged from 56.7% to 94.7% of total activity. Serum BALP activity ranged from 5.3% to 42.8%. There was negligible IALP activity in all serum samples.     

Immunological investigation of intestinal, liver, kidney, bone, placental and serum alkaline phosphatase in cattle

The purpose of this study is to investigate the immunological difference between intestinal, liver, kidney, bone, placental and serum alkaline phosphatase (ALPs) in cattle. Kidney, bone and placental ALPs were purified from each tissue by gel filtration and ion-exchange chromatographies, and intestinal, liver, and placental ALPs were obtained from a commercial source. The antibody to each tissue ALP was prepared by immunizing rabbits and tested by double immunodiffusion analysis in cross reactivities. The results indicated that the bovine tissue ALPs are divided into four groups in antigenicity, that is, intestinal, bone, liver and kidney-placental ALPs. The fetus, calf and cow sera contained bone ALP, and calf and cow sera also contained kidney or placental ALP, but all of the sera did not contain intestinal and liver ALP.     

Enzyme-linked immunosorbent assay for measurement of allergen-specific IgE antibodies in canine serum

A micro-ELISA, using horseradish peroxidase-conjugated anti-canine IgE and polystyrene microtitration wells for detection of allergen-specific IgE in canine serum, was developed. Specificity of anti-canine IgE was confirmed by reversed cutaneous anaphylaxis evaluations, gel-precipitation reactions, immunoelectrophoresis, immunoaffinity chromatography, and heat inactivation. Individual allergen blanks were used to account for variable nonspecific binding among various allergens, and results were normalized using 4 reference sera. Coefficients of variation for intra-assay and interassay variability ranged from 0.77 to 5.66% and 3.15 to 9.83%, respectively. Results observed with wells coated with mixtures of various allergen extracts yielded results approximately equal to results (average) of wells containing individual components. Agreement between ELISA and skin test results ranged from 43 to 64%, depending on allergen used.     

Subcellular location of corticosteroid-induced alkaline phosphatase in canine hepatocytes

Dogs received either 4 mg/kg of prednisone or sterile saline daily for 32 days. Serum samples were assayed every 4 days for total alkaline phosphatase (ALP) and corticosteroid-induced ALP isoenzyme (CIALP) activity. The initial and major increase of serum ALP was attributed to the liver isoenzyme of ALP (LALP), however, CIALP began to increase by day 8 and was significantly increased by day 24. Prior to treatment and on day 32, sections of liver from control and prednisone-treated dogs were stained for ALP activity after blocking the staining activity of LALP with levamisole. The staining activity of CIALP was compared to the staining activity of LALP in liver sections from control dogs and from dogs in which the bile duct was ligated. It was determined that CIALP was located in that area of the hepatocyte membranes which comprise the bile canaliculi.     

Development of a monoclonal antibody for the detection of anti-canine CD20 chimeric antigen receptor expression on canine CD20 chimeric antigen receptor-transduced T cells

Chimeric antigen receptor (CAR) CAR-T cell therapy targeting CD20 can be a novel adoptive cell therapy for canine patients with B-cell malignancy. After injection of the CAR-T cells in vivo, monitoring circulating CAR-T cells is essential to prove in vivo persistence of CAR-T cells. In this study, we developed a novel monoclonal antibody against canine CD20 CAR, whose single-chain variable fragment was derived from the our previously reported anti-canine CD20 therapeutic antibody. Furthermore, we proved that this monoclonal antibody can detect therapeutic anti-canine CD20 chimeric antibody in the serum from healthy beagle dogs injected with the therapeutic antibody for safety study. This monoclonal antibody is a useful tool for monitoring both canine CD20-CAR-T cells and anti-canine CD20 therapeutic antibody for canine lymphoma.     

Origin of serum alkaline phosphatase in the dog.

The origin of canine serum alkaline phosphatase (ALP) was investigated by various means. On the basis of electrophoretic migration, neuraminidase treatment, thermal denaturation, and chromatographic fractionation, canine serum was found to contain ALP principally of hepatic origin. There was evidence of only a minor portion of ALP being of osseous origin. Intestinal ALP was not detected in canine serum when monitored by immunochemical technique, L-phenylalanine inhibition, and thermal denaturation.     

Multiple forms of alkaline phosphatase in horse bone,liver, kidney,duodenum, caecum and serum separated by agarose gel electrophoresis with and without neuraminidase pretreatment

Horse bone, liver, duodenum, caecum and kidney alkaline phosphatases were separated by a commercial agarose gel electrophoresis method with and without neuraminidase pretreatment, following the manufacturer's directions. Tissue extracts were obtained in saline solution and ALP extracted from cell membranes by the butanol method. Electrophoresis was performed using a TRIS/barbital/sodium barbital buffer with detergent, pH 8.6 to 9.0, at 250 V for 30 minutes. Bone, liver and kidney untreated extracts showed two ALP bands each, but with different relative migration (compared to albumin migration). When they were preincubated with neuraminidase, the two bone bands showed a marked decrease in their migration, followed by the kidney ALP bands and the most anodic band of liver Both intestinal untreated extracts showed three bands but with different mobilities. After preincubation with neuraminidase, the three bands of caecum mucosa decreased in their migration, and the most anodic duodenum band disappeared, overlapping the second one. When tissue extracts were incubated with wheat germ-lectin (WGL), 74.5% of bone extract ALP and 67.2% of caecum extract ALP precipitated, which demonstrated that the ALP band of both tissues have similar groups in the carbohydrate side chains. Horse serum showed two electrophoretic bands, which increased to three bands when treated with neuraminidase. ALP from hepatocytes was the dominant isoform, followed by a caecum band. Because the electrophoretic mobilities of some of the tissue bands studied were identical, the neuraminidase agarose electrophoretic method appeared to be a satisfactory alternative to separate them.     

Diagnostic value of intestinal alkaline phosphatase in horse serum

Antiserum directed against equine intestinal Alkaline Phosphatase (ALP) was produced in rabbits and used to develop a sensitive and quantitative assay for the detection of intestinal ALP in equine serum. This assay was then used to measure the half-life of intravenously injected intestinal ALP and to determine if the intestinal ALP was present in normal horse sera, sera from horses presented for lesions not involving the gastrointestinal tract and sera from horses presented with lesions involving the gastrointestinal tract. The results suggest that intestinal ALP is not likely to appear in equine serum even when gastrointestinal disease is present and, therefore, appears to be of no diagnostic value.     

Spectrophotometric method for differentiation of cardiac and hepatic lactate dehydrogenase activities in dogs

To differentiate the origin of high total lactate dehydrogenase (LD) activity in canine sera, a spectrophotometric method based on the preferential inhibition of cardiac LD isoenzymes by pyruvate was performed. Comparison with the electrophoretic separation of LD isoenzyme activities and determination of the hydroxybutyrate dehydrogenase-to-LD ratio indicated that the method proposed gave a better discrimination between cardiac and hepatic LD activities than did the other tests.     

Measurement of canine IgE using the alpha chain of the human high affinity IgE receptor

In vitro assays for allergen specific immunoglobulin E (IgE) are a convenient and reproducible alternative to intradermal skin testing in dogs. Such tests may be used to support a diagnosis of atopic dermatitis and to define appropriate allergens for immunotherapy. Current in vitro assays rely upon monoclonal or polyclonal antibodies as IgE detection reagents. However, in sera where allergen-specific IgG occurs in great excess, any IgE:IgG cross-reactivity of the detection reagent may result in lowered assay specificity. Therefore, we have developed an assay for canine IgE which uses a recombinant form of the extracellular part of the alpha chain of the human high affinity IgE receptor (FcvarepsilonRIalpha). Biotinylated FcvarepsilonRIalpha shows no significant binding to purified canine IgG, and recognizes a heat labile antibody in serum, with a detection limit of 73-146pg/ml. Comparison of assay signals using the labeled FcvarepsilonRIalpha and a highly specific anti-canine IgE monoclonal antibody (MAb) shows good agreement. The FcvarepsilonRIalpha is therefore a sensitive and specific alternative to polyclonal or monoclonal antibodies for canine serum IgE measurement.     

Lactate dehydrogenase and its isoenzymes in the tissues and sera of clinically normal dogs

The total activity of lactate dehydrogenase (LDH) and the percentage distribution of its isoenzymes in the tissues and sera of clinically normal adult dogs are presented. Total LDH activity was greatest in skeletal muscle followed by heart muscle, kidney, small intestinal mucosa, liver, lung, pancreas and bone. Each tissue had a unique isoenzyme pattern and the proportions of the isoenzymes in serum suggested that liver is the source of normal serum LDH. The tissue isoenzyme patterns were similar to those obtained by other authors in human beings, horses, cattle, sheep and cats although in liver, differences between ruminants and monogastric animals including dogs were evident. The data presented provide a basis for the interpretation of serum LDH isoenzyme patterns in canine disease.     

Experimental Detection of Canine Haemoglobin (Occult Blood) in Canine Faeces by Reversed Passive Latex Agglutination

A reversed passive latex agglutination test (RPLA) using anti-canine haemoglobin (Hb) antibody was developed for detecting bleeding in the lower digestive organs in dogs, and its applicability as a simple test for faecal occult blood was assessed. In Ouchterlony's gel immunodiffusion test, the anti-canine Hb antibody used to sensitize the latex reacted with canine Hb but not with Hbs, plasmas or meat extracts from pigs, goats, sheep, cattle, horses or chickens, or with fish extracts. Using latex sensitized with 50 µg/mg of anti-canine Hb IgG antibody, the lowest limit of detection for canine Hb was 21 µg/ml, and the latex reacted negatively with all test specimens other than canine Hb. In an in vitro experiment with a mixture of canine faeces and erythrocytes, the antigenicity of the Hb was found to undergo only very slight changes even when the specimens were allowed to stand for 12 h at room temperature. Hb could not be detected by RPLA in any of four successive faecal samples from three experimental dogs after infusion of autologous blood (5, 3 or 1 ml) into the stomach. In 3 other experimental dogs given an infusion of autologous blood (5, 3 or 1 ml) into the ascending colon, the presence of Hb was confirmed by RPLA in all four successive faecal samples obtained from those which received 5 or 3 ml of blood and in all except that obtained following the first defecation from the animal which had received 1 ml of blood.     

Electrophoretic evaluation of sera from dogs with cancer.

A quantitative electrophoretic method was developed for evaluating sera from dogs with naturally occurring malignant tumors. Electrophoretic migration ratios (EMR) were devised to standardize characteristic protein components and to analyze alterations in mobility of these protein components, using a computerized densitometric technique. Sera values from dogs with lymphosarcomas, osteosarcomas, and mammary tumors were compared, using conventional methods of statistical analysis. Results indicate a statistical difference existed between mean values of EMR and alpha2-globulins of sera from dogs with tumors and those values from normal dogs. In addition, unique EMR and concentration of protein components were identified for dogs with lymphosarcomas, osteosarcomas, and mammary tumors.     

Effect of substrate selection on indirect immunofluorescence testing of canine autoimmune subepidermal blistering diseases

The detection by indirect immunofluorescence (IIF) of circulating antibodies in the serum of dogs with autoimmune subepidermal blistering diseases (AISBD) was regarded for a long time as an unrewarding tool. It was, however, demonstrated in humans that the sensitivity of IIF assays depended on the selection of the substrates used. The effects of substrate selection on IIF tests was thus studied by examining sera from 12 dogs with AISBD tested against 8 different substrates from 3 different normal dogs. Patients with AISBD suffered from bullous pemphigoid (n = 4 sera), mucous membrane pemphigoid (n = 4 sera), and epidermolysis bullosa acquisita (n = 4 sera). Substrates included canine tongue, canine lip, canine dorsal haired skin, and ventral haired skin. The same 4 substrates were also split with salt splitting technique (using 1 M sodium chloride), in order to cleave the basement membrane within the lamina lucida and to expose the targeted antigens. The strength of the specific fluorescence of each slide was scored after processing for IIF testing with anti-canine IgG polyclonal antibody. Other criteria, such as background fluorescence, easiness of the interpretation, and variations within a same substrate, were also assessed. Intact canine lip and canine salt-split lip demonstrated consistently stronger intensity of fluorescence and a better ease of interpretation. We concluded that the performance of IIF tests with such substrates was a reliable tool for the detection of circulating IgG autoantibodies of canine patients with AISBD.     

Alkaline phosphatase and its isoenzymes in the tissues and sera of normal dogs

The total alkaline phosphatase (AP) activity and the pattern of its isoenzymes were studied in the tissues and sera of normal adult dogs. Small intestine mucosa showed the greatest total AP activity followed by kidney, bone, pancreas, liver, lung, skeletal muscle and heart muscle. After separation by agarose gel electrophoresis, each tissue showed only one isoenzyme except lung which showed two. The tissue isoenzymes, in decreasing order of migration distance towards the anode, were as follows: fast lung isoenzyme, liver or slow lung isoenzyme, the group consisting of skeletal muscle, bone, small intestine and pancreas isoenzymes and, finally, the kidney isoenzyme. Two isoenzymes occurred in serum. The major band corresponded to liver and the slow lung isoenzyme, while the minor band was considered to be the corticosteroid-induced isoenzyme, previously thought to be absent from normal serum.The AP isoenzyme patterns in lung and skeletal muscle and the presence of an isoenzyme migrating an identical distance to the corticosteroid-induced isoenzyme do not appear to have been reported before in normal dogs.     

Alkaline phosphatase and its isoenzymes in the tissues and sera of normal dogs

The total alkaline phosphatase (AP) activity and the pattern of its isoenzymes were studied in the tissues and sera of normal adult dogs. Small intestine mucosa showed the greatest total AP activity followed by kidney, bone, pancreas, liver, lung, skeletal muscle and heart muscle. After separation by agarose gel electrophoresis, each tissue showed only one isoenzyme except lung which showed two. The tissue isoenzymes, in decreasing order of migration distance towards the anode, were as follows: fast lung isoenzyme, liver or slow lung isoenzyme, the group consisting of skeletal muscle, bone, small intestine and pancreas isoenzymes and, finally, the kidney isoenzyme. Two isoenzymes occurred in serum. The major band corresponded to liver and the slow lung isoenzyme, while the minor band was considered to be the corticosteroid-induced isoenzyme, previously thought to be absent from normal serum. The AP isoenzyme patterns in lung and skeletal muscle and the presence of an isoenzyme migrating an identical distance to the corticosteroid-induced isoenzyme do not appear to have been reported before in normal dogs.