Validation of the BioPRYN enzyme‐linked immunosorbent assay for detection of pregnancy‐specific protein‐B (PSPB) and diagnosis of pregnancy in American bison (Bison bison)

This study assessed the accuracy of the commercial BioPRYN^® ELISA for the detection of pregnancy‐specific protein‐B (PSPB) using a single blood sample to determine pregnancy status in American bison (Bison bison). A total of 49 bison cows were used in the study, and sampled at two time‐points during the gestation period, fall and spring, correlating with early‐ to mid‐term gestation (average 62.9 days post‐mating) and mid‐ to late‐term gestation (average 229.2 days post‐mating), respectively. Sensitivity of the test during early‐ to mid‐term gestation sampling period (fall) was 87.1%, while specificity was 100%; sensitivity of the test during late‐term gestation sampling period (spring) was 96.3%, while specificity remained at 100%. In total, the test showed a total sensitivity of 91.4%, specificity of 100% and total accuracy of 93.8%, similar to domestic cattle. Use of the single‐sample BioPRYN^® ELISA in American Bison for pregnancy diagnosis is economical and practical, minimizing animal handling time, frequency and subsequent stress while providing accurate results for pregnancy diagnosis at 62 days post‐mating. This method should be considered over more traditional pregnancy diagnosis methods for use in managed bison herds.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of canine sarcoptic mange

Abstract The purpose of this study was to evaluate a serodiagnostic test (enzyme-linked immunosorbent assay; ELISA) for sarcoptic mange in dogs and to characterize the assay antigen, based on the mite Sarcoptes scabiei var. vulpes. The ELISA, applied to sera from 359 dogs suspected of having sarcoptic mange, showed a sensitivity and specificity of 92 and 96%, respectively. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the antigen employed in the ELISA revealed polypeptide bands with molecular weights ranging between 14 and 164 kDa. In Western blot analyses antigens of molecular weights between 62 and 64 kDa dominated. Particularly dominant were antigens of 164 and 147 kDa. These were found to have isoelectrical points in the range of 5.7–6.9. Sera from dogs infected with Cheyletiella sp., Demodex canis, Linognathus setosus and Otodectes cynotis, as well as from dogs allergic to fleas, were negative in the ELISA. Résumé— Le but de cette étude est d'évaluer un test sérologique ELISA pour le diagnostic de la gale sarcoptique chez le chien et de caractériser l'antigène révélateur, extrait de l'acarien Sarcoptes scabiei var. vulpes. Le test ELISA, lors d'une étude conduite avec les sérums de 359 chiens suspects de gale sarcoptique a démontré une sensibilité et une spécificité de 92 et 96%, respectivement. L'électrophorèse en gel polyacrilamide dodécyl sulfate de sodium de l'antigène utilisé dans l'ELISA a révélé des bandes polypeptidiques de poids moléculaire compris entre 14 et 164 kDa. Dans l'analyse en Western blot, les antigènes de poids moléculaire compris entre 62 et 164 kDa étaient les plus abondants, notamment ceux de 164 et 147 kDa. Ces derniers ont des points isoélectriques compris entre 5.7 et 6.9. Les sérums de chiens infectés par des Cheyletiella sp. Demodex canis, Linognathus setosus et Otodectes cynotis, ou par des chiens allergiques aux puces, se sont révélés négatifs en ELISA. [Bornstein, S., Thebo, P., Zakrisson, G. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of canine sarcoptic mange (Evaluation d'un test ELISA pour le diagnostic sérologique de la gale sarcoptique canine). Veterinary Dermatology 1996; 7 : 21–28.] Resumen El objetivo de este estudio fue el de evaluar una pruba serodiagnóstica (prueba de inmunoadsorción ligada a enzima; ELISA) para la sarna sarcóptica en el perro y caracterizar el antigeno prueba, basado en el ácaro Sarcoptes scabei, var. vulpes. El ELISA, aplicado a sueros de 359 perros sospechosos de padecer sarna sarcóptica, mostró una sensibilidad y especificidad del 92 y 96%, respectivamente. La electroforesis en gel de poliacrilamida dodecil sulfato sódico (SDS-PAGE) del antigeno usado en el ELISA reveló bandas de polipétidos con peso molecular entre 14 y 164 kDa. En el análisis Western blot, predominaron los antigenos de pesos moleculares entre 62 y 164 kDa. Los antigenos entre 164 y 147 kDa fueron especialmente predominantes. Estos tuvieron puntos isoeléctricos entre 5.7 y 6.9. Los sueros de perros infectados por Cheyletiella sp., Demodex canis, Linognathus setosus y Otodectes cynotis, asi como el de perros alérgicos a las pulgas fueron negativos en el ELISA. [Bornstein, S., Thebo, P., Zakrisson, G. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of canine sarcoptic mange (Evaluation de una prueba de immunoadsorcion ligada a enzima (ELISA) para el diagnostico serologico de la sarna sarcóptica canina). Veterinary Dermatology 1996; 7 : 21–28.] Zusammenfassung— Ziel dieser Studie war, einen Serodiagnostiktest (Enzyme-Linked-Immunosorbent-Assay, ELISA) für Sarkoptesräude des Hundes zu überprüfen und das Testantigen zu charakterisieren, das auf der Milbe Sarcoptes scabiei var. vulpes basiert. Der ELISA-Test, der bei den Sera von 359 Hunden mit Sarkoptesverdacht angewendet wurde, zeigte eine Sensitivität von 92% bzw. 96%. Die Natriumdodecylsul-fatpolyacrylamid-Gelelektrophorese (SDS-PAGE) des Antigen, das im ELISA verwendet wurde, zeigte Polypeptid-Banden mit Molekulargewichten zwischen 14 und 164 kDa. In der Wester-blot-Analyse dominierten Antigene mit einem Molekulargewicht zwischen 62 und 164 kDa. Besonders dominierend waren Antigene von 164 und 147 kDa. Bei diesen stellte man isoelektrische Punkte im Bereich von 5,7 bis 6,9 fest. Die Sera von Hunden, die mit Cheyletiella sp., Demodex canis, Linognathus setosus und Otodectes cynotis infiziert waren, fielen ebenso wie die Hunde mit Allergie auf Flöhe im ELISA negativ aus. [Bornstein, S., Thebo, P., Zakrisson, G. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of canine sarcoptic mange (Die Auswertung eines Enzym-Linked-Immunosorbent-Assay (ELISA) für die serologische Diagnose der kaninen Sarkoptesräude). Veterinary Dermatology 1996; 7 : 21–28.]     

Validation of a commercially available enzyme-linked immunosorbent assay (ELISA) for the determination of cortisol in canine plasma samples

Samples were obtained from clinically normal dogs before and after ACTH stimulation and dexamethasone suppression tests. The test kit Enzymun-Test (Boehringer Mannheim) for determining cortisol concentrations in human plasma was used in connection with the analyser system Enzymun-Test (Boehringer Mannheim) System ES300 following the manufacturer's instructions. The intra-assay and inter-assay coefficients of variation were 1.28% and 5.64%, respectively. The mean recovery when assaying samples with a cortisol content of more than 100 nmol/L was 95.41%, but this percentage decreased in samples with lower cortisol levels. The sensitivity of the assay was 2.76 nmol/L. The results of the ACTH stimulation and dexamethasone suppression tests were similar to those published previously. The ELISA method evaluated allows a precise and sensitive determination of cortisol concentrations in canine plasma samples. The major drawback observed was the loss of accuracy at low cortisol concentrations. Since the assay tends then to report lower cortisol concentrations, the generally accepted concentration of 40 nmol/L may not be suitable as the cutoff value in dexamethasone suppression tests.     

Ovine sperm DNA oxidation quantification using an 8‐OHdG immunodetection assay

Our aim was to optimize 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) immunodetection in order to detect DNA damage caused by oxidative stress that may not be detected by other DNA integrity analysis techniques, especially due to the high compaction of DNA in ruminants. Semen samples from 6 rams were cryopreserved. After thawing, samples were subjected to the DNA oxidation quantification using an 8‐OHdG immunodetection assay by flow cytometry. We have evaluated two different incubation times (30 min vs. overnight) at 4°C of the primary antibody (monoclonal anti‐8‐OHdG antibody). We have also compared the results of this technique with the sperm chromatin structure assay (SCSA^®). The analysis revealed that there were no significant differences (p > .05) between different incubation times. However, overnight incubation seems to cause more non‐specific binding of the secondary antibody. Significant differences (p < .05) between subjects and oxidation controls (8 M H₂O₂/800 μM FeSO₄?7H₂O) were evident. We can conclude that the 8‐OHdG immunodetection assay for DNA oxidation quantification of ram sperm can be performed subjecting sperm samples to a very high oxidative treatment.     

Enzyme linked immunosorbent assay (ELISA) for the detection of antibodies to porcine cytomegalovirus.

The ELISA and indirect immunofluorescence test were compared on 56 porcine sera which were tested for antibodies to porcine cytomegalovirus. Viral antigens were prepared in cells of a pig fallopian tube line. The ELISA was found to be a sensitive reproducible and practical test to measure specific antibodies to this infection.     

Development and analytical validation of an enzyme linked immunosorbent assay for the measurement of canine gastric lipase immunoreactivity in serum

The objective of this study was to develop and analytically validate an enzyme linked immunosorbent assay (ELISA) for measurement of canine gastric lipase immunoreactivity (cGLI). A sandwich ELISA was developed using canine gastric lipase (cGL) purified from canine stomachs and polyclonal antibodies directed against cGL, raised in rabbits and purified by affinity chromatography. The assay was validated by determination of sensitivity, working range, linearity, accuracy, precision, reproducibility, and the upper limit of the control range by determining the 97.5th percentile of serum cGLI concentration in 74 healthy canines. Sensitivity and working range in serum were 200 ng/L and 200 to 39 160 ng/L, respectively. Observed to expected ratios for dilutional parallelism for 3 serum samples and 3 dilutions ranged from 86.1% to 244.2% (mean +/- standard deviation [s]; 125.4% +/- 48.2%). Observed to expected ratios for spiking recoveries for 3 serum samples and 6 spiking concentrations ranged from 66.4% to 152.5% (mean +/- s; 104.5% +/- 22.9%). Intra-assay and interassay variabilities for 3 different serum samples were 25.5%, 9.4%, and 13.4% and 26.0%, 17.2%, and 14.4%, respectively. The upper limit of the control range for serum cGLI was 662 ng/L. We concluded that the ELISA for cGLI described here is highly sensitive and shows a wide working range. However, the validation characteristics for this assay are suboptimal and below values of approximately 2.000 ng/L the assay is more semiquantitative in nature. Despite its limitations, whether this assay is useful for the diagnosis of canine gastric disorders remains to be determined.     

Enzyme-linked immunosorbent assay (ELISA) using staphylococcal protein A for the measurement of rabies antibody in various species

An ELISA was developed using staphylococcal protein A linked with horseradish peroxidase for detecting IgG antibody of rabies virus in human and carnivore sera (80 human, 270 fox, 40 cat, 35 marten, 5 badger and 4 polecat sera were tested in the present work). In comparison with the serum neutralization (SN) test in cell culture, close overall agreement was obtained particularly in human and cat sera (97.5%). Two post-vaccination human sera were found positive with ELISA values of 2.16 and 2.65 IU/0.2 ml, but with SN titers less than 1:10. All prevaccination human sera were found negative by both tests. Regression analysis on 30 post-vaccination human sera revealed better correlation between ELISA and SN test at a serum dilution of 1:100 than at lower serum dilutions of 1:80 or 1:20. The correlation coefficient (r) was 0.73 (p less than or equal to 0.0001).     

Development of an enzyme linked immunosorbent assay (ELISA) for the detection of bovine serum antibody to bovine viral diarrhoea virus

An enzyme linked immunosorbent assay (ELISA) was developed to detect antibody to bovine viral diarrhoea virus (BVDV) in bovine serum. The ELISA results were compared with those of the serum neutralisation test (SNT) using serums from 6 experimentally infected calves bled at intervals from 0 to 154 days postinfection and 886 field samples. The optical density (OD) produced by a single dilution of test serum was compared with a standard curve and the result expressed in ELISA units. Despite wide variation between absolute ELISA and SNT results, an agreement of 97% was obtained when reciprocal SNT titres greater than or equal to 8 and ELISA units greater than or equal to 10 were taken as indicative of a specific reaction. The ELISA was shown to be an efficient method of measuring antibody in bovine serum samples and would assist in any large scale screening of cattle herds for BVDV antibody.