Objective—To develop and evaluate a sandwich ELISA incorporating rabbit antiserum specific for canine surfactant protein A (SP-A) for use in measuring concentrations of SP-A in serum of dogs.
Sample—Serum samples obtained from 6 healthy dogs and 3 dogs with pulmonary disease.
Procedures—Rabbit antiserum was prepared against purified canine SP-A. The IgG fraction was isolated via protein G affinity chromatography and was then biotinylated. The sandwich ELISA was performed by use of anti-SP-A antibody (IgG) preabsorbed with sera from healthy dogs. Validity of the ELISA was confirmed by determination of the detection limit, precision, reproducibility, and accuracy. Serum SP-A concentrations were measured in 6 healthy dogs and 3 dogs with pulmonary disease.
Results—Detection limit of the ELISA was 2.0 ng/mL. Within- and between-assay coefficients of variation ranged from 3.8% to 14.1% and from 15.5% to 35.6%, respectively. The observed-to-expected recovery ratio ranged from 77.1% to 89.9%. Serum SP-A concentrations measured by use of the ELISA were ≤ 2.3 ng/mL in the 6 healthy dogs, 25.6 ng/mL in a dog with severe cardiac pulmonary edema, 8.3 ng/mL in a dog with pneumonia, and 10.1 ng/mL in a dog with lung lobe torsion.
Conclusions and Clinical Relevance—The sandwich ELISA was found to be useful for measuring purified canine SP-A concentrations and canine SP-A concentrations in serum samples. The ELISA was precise, reproducible, and accurate. The ELISA may be beneficial in assessing serum concentrations of canine SP-A as a potential biomarker of pulmonary diseases in dogs.
To assess whether the volume of extruded materials is correlated with neurologic severity in dogs with type I thoracolumbar intervertebral disk herniation (TL-IVDH).
70 client-owned small-breed dogs with type I TL-IVDH diagnosed between July 1, 2016, and June 30, 2018.
For this retrospective cohort study, the medical records of 70 dogs with surgically confirmed type I TL-IVDH were reviewed. The volume and height of the intervertebral disk and the area of the maximal transverse compressed spinal cord were measured using CT myelographic images. For each dog, the volume of the disk immediately cranial to the herniated disk was an internal control. Dogs were grouped on the basis of grade of neurologic severity.
Preoperative grades of neurologic severity were grade 2 in 7 (10%) dogs, grade 3 in 16 (23%) dogs, grade 4 in 28 (40%) dogs, and grade 5 in 19 (27%) dogs. The total volume of the affected intervertebral disks was significantly larger than the internal control. Weak positive correlation was found between the volume of the extruded materials into the vertebral canal and the grade of neurologic severity.
Our findings indicated that the total volume of the affected intervertebral disks is larger in dogs with type I TL-IVDH, and the volume of the extruded materials into the vertebral canal is weakly correlated with the neurologic severity.
Objective—To determine whether cross-reactivity
exists between canine chromogranin A (CgA) and
anti-human CgA antibody and investigate the usefulness
of plasma CgA concentration measurements as
an index of acute stress responses in dogs.
Animals—12 healthy Beagles.
Procedure—Canine CgA was extracted and purified
from canine adrenal glands of cadaver dogs for studying
cross-reactivity with anti-human CgA antibody.
Western blotting with anti-human CgA antibody was
performed. Blood samples were collected from dogs
at 0, 10, 20, 30, 40, 60, 120, and 180 minutes after IV
administration of saline (0.9% NaCl) solution or
insulin. Canine plasma CgA concentrations were
determined by use of a CgA ELISA kit with rabbit antiserum
against the carboxy-terminal fragment of
human CgA. Plasma cortisol and catecholamine (ie,
norepinephrine and epinephrine) concentrations were
measured by use of an ELISA and a high-performance
liquid chromatography method, respectively.
Results—Purified canine CgA was specifically detected
by use of western blot analysis and an ELISA with
anti-human CgA antibody. An increase in plasma CgA
concentrations was observed in insulin-induced hypoglycemic
dogs. Changes in plasma CgA concentration
were correlated with changes in plasma cortisol or
catecholamine concentrations of hypoglycemic dogs.
Conclusions and Clinical Relevance—Use of the
CgA ELISA kit for determination of human plasma
CgA concentrations is applicable to the measurement
of canine plasma CgA concentrations. Canine plasma
CgA concentrations, along with measurements of
plasma cortisol and catecholamine concentrations,
correctly reflect insulin-induced hypoglycemic
stressed conditions in dogs. Measurement of canine
plasma CgA concentrations may provide a useful
index for evaluation of an acute stress response. (Am
J Vet Res 2005;66:1830–1835)
To compare ultracentrifugation, precipitation, and membrane affinity chromatography methods for isolation of extracellular vesicles (EVs) from canine plasma samples and to identify suitable reference genes for incorporation into a quantitative reverse transcription PCR assay of microRNA expression in plasma EVs of healthy dogs.
6 healthy Beagles.
Plasma samples were obtained from each dog, and EVs were isolated from 0.3 mL of these samples via ultracentrifugation, precipitation, and membrane-affinity chromatographic methods. Nanoparticle tracking analysis was performed to determine the concentration and size distribution of EVs isolated by the ultracentrifugation method. Expression levels (cycle threshold values) of 4 microRNAs (let-7a, miR-16, miR-26a, and miR-103) were then compared by means of quantitative reverse transcription PCR assay. Three statistical programs were used to identify the microRNAs most suitable for use as reference genes.
Results indicated that ultracentrifugation was the most stable of all 3 methods for isolating microRNAs from 0.3 mL of plasma. Nanoparticle tracking revealed that EV samples obtained by the ultracentrifugation method contained a mean ± SD of approximately 1.59 × 1010 vesicles/mL ± 4.2 × 108 vesicles/mL. Of the 4 microRNAs in plasma EVs isolated by ultracentrifugation, miR-103 was the most stable.
CONCLUSIONS AND CLINICAL RELEVANCE
The ultracentrifugation method has potential as a stable method for isolating EVs from canine plasma samples with a high recovery rate, and miR-103 may provide the most stable reference gene for normalizing microRNA expression data pertaining to plasma EVs isolated by ultracentrifugation.
To measure expression of microRNAs (miRNAs) in plasma and in extracellular vesicles (EVs) derived from plasma for dogs with glioma and dogs with other brain diseases.
Plasma samples from 11 dogs with glioma and 19 control dogs with various other brain diseases.
EVs were isolated from plasma samples by means of ultracentrifugation. Expression of 4 candidate reference miRNAs (let-7a, miR-16, miR-26a, and miR-103) and 4 candidate target miRNAs (miR-15b, miR-21, miR-155, and miR-342-3p) was quantified with reverse transcription PCR assays. Three software programs were used to select the most suitable reference miRNAs from among the 4 candidate reference miRNAs. Expression of the 4 target miRNAs was then calculated relative to expression of the reference genes in plasma and EVs, and relative expression was compared between dogs with glioma and control dogs with other brain diseases.
The most suitable reference miRNAs were miR-16 for plasma and let-7a for EVs. Relative expression of miR-15b in plasma and in EVs was significantly higher in dogs with glioma than in control dogs. Relative expression of miR-342-3p in EVs was significantly higher in dogs with glioma than in control dogs.
CONCLUSIONS AND CLINICAL RELEVANCE
Results suggested that miR-15b and miR-342-3p have potential as noninvasive biomarkers for differentiating glioma from other intracranial diseases in dogs. However, more extensive analysis of expression in specific glioma subtypes and grades, compared with expression in more defined control populations, will be necessary to assess their clinical relevance.