Development and analytical validation of an enzyme-linked immunosorbent assay for the measurement of feline tumor necrosis factor α in serum.

BACKGROUND: The role of tumor necrosis factor alpha (TNF-α), a cytokine shown to play a crucial role in human Crohn's disease patients, has not been documented in cats with chronic enteropathies. Also, currently, no validated assay for measurement of TNF-α in cats is available. OBJECTIVES: The objective of this study was to develop and analytically validate an enzyme-linked immunosorbent assay (ELISA) for the quantification of TNF-α in serum from cats. METHODS: A sandwich ELISA was developed and analytically validated by assessment of detection limit, linearity, accuracy, precision, and reproducibility. A control range for serum fTNF-α concentration in healthy cats was established. In addition, serum concentrations of fTNF-α in 39 cats with chronic enteropathies were compared with those in 20 healthy cats. RESULTS: The detection limit of the assay was 38.4 ng/L. Observed-to-expected ratios for serial dilutions of 4 serum samples ranged from 75.1% to 111.9%. Observed-to-expected ratios for spiking recovery for 4 serum samples ranged from 91.3% to 129.7%. Coefficients of variation for intra- and inter-assay variability ranged from 3.9% to 7.6% and from 7.8% to 12.5%, respectively. The control range of the assay was < 38.4-223.5 ng/L. Serum concentrations of feline TNF-α were significantly higher in cats with chronic enteropathies and diarrhea than in cats with chronic enteropathies without diarrhea, or in healthy control cats. CONCLUSIONS: The ELISA described here was suitable for the quantification of fTNF-α in feline serum and should facilitate research into the importance of TNF-α in cats with chronic enteropathies.

Effect of syringe and aggregate filter administration on survival of transfused autologous fresh feline red blood cells.

OBJECTIVE: To assess the effect of transfusion using a syringe and microaggregate filter on short-term survival and circulating half-life of autologous feline RBCs. DESIGN: Prospective, internally controlled, observational study. SETTING: A University Teaching Hospital ANIMALS: Six apparently healthy, owned cats. INTERVENTIONS: Blood collection by jugular venipuncture. Transfusion with labeled, autologous, fresh RBCs. MEASUREMENTS AND MAIN RESULTS: Anticoagulated whole blood (35 mL/cat) was collected in 2 equal aliquots. RBCs were washed and labeled at 2 different biotin densities, before suspension in autologous plasma. Labeled RBCs were then transfused using 2 methods, gravity flow and pump delivery using a 20 mL syringe and 18 µm microaggregate filter. Whole blood samples were collected from each cat at 2-hour intervals for 12 hours following completion of the transfusions. Additional samples were collected at weekly intervals up to 6 weeks to assess circulating half-life of the transfused cells. Cell survival was assessed via flow cytometry. The proportion of transfused cells remaining in each of the 2 populations was measured. Biotinylated RBCs were readily detected in all cats over the 6-week sampling period. There was a significant decrease in both populations of labeled cells over the 6-week period (P < 0.01), as expected. There was no difference in probability that the RBCs would survive up to 12 hours immediately following transfusion, and no significant difference in survival between the 2 groups over 6 weeks. The average half-life of all labeled cells was approximately 23 days. CONCLUSIONS: We conclude that, in contrast to findings from dogs, transfusion of autologous feline RBCs using a syringe + aggregate filter method does not significantly impact short- or long-term survival of the transfused cells.

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts.

BACKGROUND: Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. RESULTS: Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. CONCLUSIONS: The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.

Post-transfusion survival of biotin-labeled allogeneic RBCs in adult horses.

BACKGROUND: Post-transfusion survival of allogeneic RBCs has been reported to be much shorter in horses than in other species. We hypothesized that post-transfusion survival of biotinylated allogeneic equine RBCs would be greater than the survival previously reported from studies using radioactive RBC-labeling techniques. OBJECTIVE: The study objective was to determine post-transfusion survival of N-hydroxysuccinimide (NHS)-biotin-labeled allogeneic equine RBCs transfused into adult horses. METHODS: Horses were adults and included 5 donors and 5 recipients. All horses were blood-typed, and donors were paired with recipients based upon blood type and crossmatch results. Donor blood was collected in a volume of 4 L into citrate phosphate dextrose adenine-1 and stored for 24 hours, labeled with NHS-biotin, and re-infused into recipients. Post-transfusion blood samples were collected at 15 minutes and at 1, 2, 3, 5, 7, 14, 21, 28, and 35 days. Biotin-labeled RBCs were detected by flow cytometry using streptavidin-phycoerythrin. Post-transfusion survival at 24 hours, lifespan, and half-life of biotinylated RBCs were determined. RESULTS: Mean ± SD survival of biotinylated RBCs at 24 hours post-transfusion was 95 ± 24%; the mean lifespan of transfused allogeneic RBCs was 39 days based on calculation of a linear regression survival curve, and mean post-transfusion RBC half-life was 20 days. CONCLUSIONS: Post-transfusion survival of 24-hour stored equine allogeneic RBCs was greater than previously reported but less than that observed for other companion animal species. Mechanisms for the relatively short post-transfusion lifespan of allogeneic equine RBCs remain unknown and warrant further study.

Influence of transfusion technique on survival of autologous red blood cells in the dog.

OBJECTIVE: To determine the effect of 3 differing transfusion techniques on survival of autologous canine RBCs. DESIGN: Prospective, blinded study. SETTING: University Teaching Hospital. ANIMALS: Nine healthy dogs. INTERVENTIONS: Three distinct preparations of RBCs, each representing ~1% of red cell mass, were generated for each dog by biotinylation of RBCs at varying biotin densities. Labeled cells were transfused using 3 techniques (gravity, volumetric pump, syringe pump). Serial determinations of red cell survival were carried out by flow-cytometric analysis of RBCs collected at 7-day intervals for 49 days. In vitro analysis of the effect of transfusion methods on RBC integrity and osmotic fragility were carried out in 7/9 dogs. MEASUREMENTS AND MAIN RESULTS: RBCs administered via volumetric and syringe pumps exhibited a marked decrease in short-term probability of survival compared with RBCs delivered by gravity flow. At 24 hours, only 4/8 and 1/7 dogs had surviving cell populations delivered by volumetric and syringe pump, respectively, compared with 8/8 dogs which had surviving cell populations delivered by gravity flow. Circulating half-life of cells surviving at 24 hours after delivery by volumetric pump was not significantly different to that delivered by gravity flow. No significant effect on in vitro RBC integrity or osmotic fragility was detected in relation to transfusion technique. CONCLUSIONS: Delivery of autologous canine RBCs via mechanical delivery systems was associated with a high risk for early loss of transfused cells.

Erythrocyte survival time in Greyhounds as assessed by use of in vivo biotinylation.

OBJECTIVE: To determine erythrocyte survival time in Greyhounds. ANIMALS: 6 Greyhounds used as blood donors and 3 privately owned non-Greyhound dogs. PROCEDURES: In vivo biotinylation of erythrocytes was performed by infusion of biotin-N-hydroxysuccinimide into each dog via a jugular vein catheter. Blood samples were collected 12 hours later and then at weekly intervals and were used to determine the percentage of biotin-labeled erythrocytes at each time point. Erythrocytes were washed, incubated with avidin-fluorescein isothiocyanate, and washed again before the percentage of biotinylated erythrocytes was measured by use of flow cytometry. Survival curves for the percentage of biotinylated erythrocytes were generated, and erythrocyte survival time was defined as the x-intercept of a least squares best-fit line for the linear portion of each curve. RESULTS: The R2 for survival curves ranged from 0.93 to 0.99 during the first 10 weeks after infusion of erythrocytes. Erythrocyte survival time for the 3 non-Greyhound dogs was 94, 98, and 116 days, respectively, which was consistent with previously reported values. Erythrocyte survival time for the 6 Greyhounds ranged from 83 to 110 days (mean, 93 days; median, 88 days). As determined by use of in vivo biotinylation, erythrocyte survival times in Greyhounds were similar to those determined for non-Greyhound dogs and did not differ significantly from erythrocyte survival times reported previously for non-Greyhound dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Erythrocyte survival time was similar in Greyhounds and non-Greyhound dogs. Greyhounds can be used as erythrocyte donors without concerns about inherently shorter erythrocyte survival time.

Use of an in vitro biotinylation technique for determination of posttransfusion survival of fresh and stored autologous red blood cells in Thoroughbreds.

OBJECTIVE: To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days. ANIMALS: 13 healthy adult Thoroughbreds. PROCEDURES: Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfused or stored at 4 degrees C for 1, 14, or 28 days prior to labeling with NHS-biotin and reinfusion. Posttransfusion blood samples were collected 15 minutes and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after reinfusion. Biotin-labeled RBCs were detected via flow cytometry by use of streptavidin-PE. Posttransfusion lifespan of RBCs and RBC half-life were determined. RESULTS: Optimal biotin concentration was 0.04 pg of biotin/RBC, and the optimal streptavidin-PE ratio was 1.2 microg of streptavidin-PE/1 x 10(6) RBCs. Posttransfusion lifespan of autologous RBCs was 99, 89, 66, and 59 days after storage for 0, 1, 14, and 28 days, respectively. Storage did not result in significant alterations in RBC lifespan. Mean posttransfusion RBC half-life was 50, 45, 33, and 29 days for 0, 1, 14, and 28 days of storage, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Biotin can be used to label equine RBCs for RBC survival studies. Posttransfusion survival of equine autologous RBCs was greater than previously reported.

Immunolocalization of cytoskeletal components in the tegument of the 3-week-old juvenile and adult Fasciola gigantica.

Components of three cytoskeletal elements, namely, microtubule, intermediate and actin filaments have been localised in the tegument of the 3-week-old juvenile and adult Fasciola gigantica by means of immunofluorescence and immunoperoxidase techniques, using mouse monoclonal anti-alpha-tubulin, anti-cytokeratin antibodies and biotinylated-phalloidin, respectively. The immunostaining with the above probes were also performed in adult Schistosoma mansoni for comparison. The presence of tubulin, indicative of microtubules, was demonstrated in the tegumental cell bodies, their cytoplasmic processes, and the basal layer of the tegumental syncytium of F. gigantica. While in S. mansoni, tubulin appeared as vertical lines stretching across the whole thickness of the syncytium. Cytokeratin, representing one type of intermediate filaments, was detected in the tegumental cell bodies, their cytoplasmic processes, tegumental syncytium and spines of F. gigantica. In contrast, cytokeratin was evident only in the syncytium of S. mansoni, but not in the spines. Phalloidin, which could bind to actin, a subunit of microfilament, was detected in the tegumental cell bodies, their processes, and the microtrabecular network which form the scaffold of the tegumental syncytium of F. gigantica. In S. mansoni, actin was localized in similar tissues except the syncytium was not stained while spines exhibited intense staining. In F. gigantica, the presence of microtubules and actin filaments in the tegumental cells, their processes and in the syncytium could mediate the movement of secretory granules from the cell bodies towards the basal as well as the apical layer of the tegument. Cytokeratin filaments may serve to reinforce the integrity of the tegumental syncytium as well as the spines.

Validation of an experimental strategy for studying surface-exposed proteins involved in porcine sperm-oviduct contact interactions.

Previous experiments have shown that boar sperm survival in vitro is enhanced when co-incubated with a solubilised protein extract of porcine oviducal apical plasma membrane proteins. Here, we examine the hypothesis that the effects are mediated by direct oviduct-sperm contact and use in situ biotinylation of the oviducal epithelial surface to trace the surface-exposed biotinylated proteins through purification and solubilisation steps. We have also examined the effectiveness of mechanical scraping as a method of recovering oviducal epithelial proteins. We show that a subset of proteins originally exposed at the oviducal surface eventually bind to spermatozoa during incubation in vitro, but also show that a different protein subset is implicated if the sperm incubation is performed with proteins that had been biotinylated after (ex situ) extraction from the oviduct. Apical plasma membrane fractions biotinylated after purification contained many more biotinylated protein bands than preparations labelled before purification and multiple protein bands were eventually found to associate with spermatozoa. Although the evidence presented here supports the hypothesis that protein(s) anchored to the oviducal epithelium bind populations of spermatozoa directly and may have a role in the enhancement of sperm viability, it also shows that the choice of investigative technique exerts a major influence on experimental outcomes.

Changes in platelet life span in dogs with mitral valve regurgitation.

Platelet life span was measured in dogs with experimentally-induced mitral regurgitation (MR) by using an in vitro whole-blood biotinylation technique to evaluate the effects of shear stress induced by MR. Mitral regurgitation was created in healthy dogs by ablation of the mitral valve chordae tendineae. Mitral regurgitation was identified by auscultation and postoperative color Doppler echocardiography. Platelets were biotinylated. and derivatized blood was reinfused into the dogs. Biotinylated platelet disappearance was measured over time to determine platelet life span. Pre- and postablation platelet life span was compared in each dog. Platelet life span was shorter in dogs with experimentally induced MR. Shear stress from MR was postulated to shorten platelet life span. Alternations in platelet function and life span may contribute to the progression of cardiovascular disease in dogs with MR. Further studies are required to determine if platelet dysfunction is related to disease progression in these patients.

Changes in serum ovotransferrin levels in chickens with experimentally induced inflammation and diseases.

A competitive enzyme immunoassay was developed to measure the changes in serum levels of ovotransferrin (OTF) during inflammation and infectious diseases in chickens. The assay is based on the competition of serum OTF with a fixed concentration of biotin-labeled OTF to bind to a rabbit anti-chicken transferrin antibody immobilized on microtiter wells. After several washing steps, the antibody-bound biotinylated OTF is probed with streptavidin-horseradish peroxidase conjugate (HRP) followed by a colorimetric detection of the HRP activity. The relative changes in the optical density of color are plotted against the competing concentrations of OTF with logarithmic regression to generate a standard curve that is used to determine the concentrations of OTF in unknown samples. Serum had no effect on the measurement of OTE By this method, the time course changes of serum OTF levels in 4-wk-old male broiler chickens that were subjected to inflammation by croton oil injection were measured. The results showed croton oil-induced inflammation elevated serum OTF levels at 16 hr postinjection. OTF levels reached a peak by 72 hr, remained high through 120 hr, and returned to a basal level of olive oil-injected controls by 240 hr. There were no changes in serum OTF levels at any of the above time points in olive oil-injected control chickens. For studies with poultry diseases, specific-pathogen-free (SPF) male chickens were challenged with known bacterial and viral pathogens, and serum was collected at the height of the infection, i.e., 7 days after the challenge. Compared with uninjected controls, the SPF chickens challenged with Escherichia coli, fowl poxvirus, respiratory enteric orphan virus, infectious bursal disease virus, infectious bronchitis virus, or infectious laryngotracheitis virus had higher levels of OTF in serum. Inflammation-induced changes in serum OTF levels were also evident in the changes in the density of a 65-kD band protein corresponding to OTF. These results demonstrate that serum OTF may be a nonspecific clinical marker of inflammation associated with traumatic or infectious avian diseases.