RESUMEN
INTRODUCTION: Endometritis and endometrosis have been and still are the major reasons for infertility in mares. The diagnosis of endometritis can be based on cytology and microbiology, but endometrial biopsy is still the only way to diagnose endometrosis in the mare. Our study attempted to determine if a single biopsy using his-topathology and immunohistochemistry is sufficient to ascertain reasons for infertility in Icelandic mares. The objectives of this study were to examine the relationship between deviations in endometrial biopsies in terms of prostaglandin-endoperoxide synthase-2 (PTGS-2) and fibronectin expression and polymorphonuclear cells (PMNs) infiltration, as well as scoring degeneration in two endometrial biopsies. MATERIAL AND METHODS: Materials were collected from 53 Icelandic breed mares, from whom two endometrial biopsies were collected and they were used for histopathology and for immunohistochemistry for PTGS-2 and fibronectin. RESULTS: In our study, twenty-six of 53 mares (49%) showed differences in the biopsy score between the left and the right uterine horns (p = 0.002). There were statistically significant differences in fibronectin expression (p = 0.001), as well as in PTGS-2 expression in the superficial epithelium (p = 0.017). CONCLUSIONS: Significant differences in the biopsy score, and fibronectin and PTGS-2 expression, between two endometrial biopsies obtained from individual mares demonstrated that a single biopsy could be insufficient for diagnosing uterine health status in Icelandic mares.
Asunto(s)
Biopsia/normas , Endometritis/veterinaria , Endometrio/patología , Enfermedades de los Caballos/diagnóstico , Animales , Biopsia/estadística & datos numéricos , Endometritis/diagnóstico , Endometrio/cirugía , Femenino , Fibronectinas/metabolismo , Fibronectinas/normas , Caballos , Inmunohistoquímica , Reproducibilidad de los ResultadosRESUMEN
A novel detection principle applicable for sensitive measurement of molecules of biological interest by time-resolved fluorescence spectrophotometry is described. Our method is based on the quantification of the Eu3+ chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) in solution in presence of an excess of Eu3+ ions. BCPDA-labeled solid phase complexes obtained by conventional immunoassay procedures are transferred into solution using urea/SDS/Eu3+ as dissociating and fluorescent lanthanide ion reagent. Two 'sandwich-type' assay variants based on the above methodology were realized for the determination of small amounts of fibronectin (FN) in biological fluids. FN is captured from solution by solid phase coated gelatin or a monoclonal antibody, respectively. Rabbit anti-FN antiserum used as second antibody is detected with a biotinylated anti-rabbit IgG antibody. Fluoresence is measured after incubation with streptavidin-BCPDA and dissociation of solid phase complexes as described. Both assays have a detection limit (blank + 3 x SD) of less than 0.5 ng/ml FN, a dynamic range of up to 300 ng/ml, and intraserial coefficients of variation of 4.4 and 6.3%, respectively. Median FN concentrations in saliva of healthy individuals were 104 (gelatin) and 36 ng/ml (double antibody), respectively.
Asunto(s)
Fibronectinas/análisis , Fluoroinmunoensayo/métodos , Quelantes , Europio , Fibronectinas/normas , Colorantes Fluorescentes , Fluoroinmunoensayo/normas , Fluoroinmunoensayo/estadística & datos numéricos , Humanos , Fenantrolinas , Estándares de Referencia , Valores de Referencia , Saliva/química , Sensibilidad y Especificidad , SolucionesRESUMEN
Fibronectin has been purified by gelatin-Sepharose affinity chromatography from fresh frozen human plasma. The bound fibronectin was eluted with 3 M urea. The purity of the fibronectin obtained has been checked on (immunoelectrophoresis, polyacrylamide gel electrophoresis, FPLC). Biological activity of the purified molecule has been monitored by means of three assays: quantitation of the gelatin-binding activity by ELISA, quantitation of the fibronectin-mediated attachment of fibroblasts on plastic and evaluation of the opsonic activity (uptake of gelatin latex particles by a murine macrophage line). When deep-frozen, fibronectin retains all of its properties. This highly purified and functional fibronectin fulfills the basic requirements for a standard reagent. It will allow to investigate physicochemical and functional alterations of various fibronectins.
Asunto(s)
Fibronectinas/aislamiento & purificación , Adhesión Celular/efectos de los fármacos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Fibronectinas/farmacología , Fibronectinas/normas , Gelatina/metabolismo , Humanos , Inmunoelectroforesis Bidimensional , Pruebas de Fijación de Látex , Proteínas Opsoninas/metabolismoRESUMEN
Several companies have developed commercial kits to measure plasma fibronectin rapidly and inexpensively with readily available laboratory equipment. In two of these kits (Cooper Biomedical and Boehringer-Mannheim) an immunoturbidimetric method is used. In a third kit (Biomedical Technologies, Inc.) an enzyme immunoassay method is used. To evaluate these commercial kits for fibronectin assay, we selected nephelometry as a comparison method for ranking the kits with regard to precision and accuracy. We also compared antibody and fibronectin cross reactivity. The antibodies from various manufacturers appear similar, but the fibronectin standards from different sources showed significant variation. Rate nephelometry and the Boehringer-Mannheim kit had the best within-run precision (CVs of 0.38% and 5.5% respectively). Between-run precision for nephelometry was excellent (CV = 1.9%) and somewhat high for the Boehringer-Mannheim kit (CV = 15.4%). This study demonstrates a need for further standardization of antigen (fibronectin) and antibody in commercial kits and the development of suitable stable quality-control material.
Asunto(s)
Fibronectinas/sangre , Juego de Reactivos para Diagnóstico/normas , Anticuerpos/normas , Reacciones Cruzadas , Fibronectinas/normas , Humanos , Inmunoensayo/métodos , Técnicas para Inmunoenzimas , Nefelometría y Turbidimetría/métodos , Control de CalidadRESUMEN
Plasma fibronectin concentrations of 148 normal canine samples were measured by rocket immunoelectrophoresis. Electrophoresis was accomplished, using 2.0% rabbit anticanine fibronectin by volume in 0.7% agarose in buffer. Films were electrophoresed 18 hours in barbital buffer, 7.5 mA/film. The mean fibronectin concentration for normal citrated dog plasma was 290 micrograms/ml +/- 50 micrograms/ml.
Asunto(s)
Fibronectinas/sangre , Inmunoelectroforesis/métodos , Animales , Perros , Fibronectinas/normasRESUMEN
A solid-phase radioimmunoassay system to quantitate human fibronectin has been developed. This assay utilizes 125I-labeled affinity purified IgG directed against human plasma fibronectin. The sensitivity of this system is comparable to conventional (labeled antigen) radioimmunoassays having a detection limit of approximately 0.5 ng. This assay is relatively rapid (less than 24 h), the reagents are stable at 4 degrees C (less than 2 months), and the reproducibility is excellent. Both human plasma and cellular fibronectin react equivalently in this assay.