ABSTRACT
The abundance of proteins in human urine is low and easily to be masked by high-abundance proteins during mass spectrometry analysis. Development of efficient and highly selective enrichment methods is therefore a prerequisite for achieving deep coverage of urine protein markers. Notably, different experimental methods would affect the urine protein enrichment efficacy and the coverage of urine proteome. In this study, ultrafiltration, nitrocellulose membrane enrichment and saturated ammonium sulfate precipitation were used to process 10 mL urine samples from five healthy volunteers and five bladder cancer patients. The urine proteins were enriched and separate by SDS-PAGE to compare the purification efficiency of different methods. Moreover, the peptide identification effects of different purification methods were analyzed by mass spectrometry to determine the best method for enriching urine protein histones. Saturated ammonium sulfate precipitation method outperformed the ultrafiltration and the nitrocellulose membrane enrichment methods in terms of the protein enrichment efficacy and quality. The interference of highly abundant albumin was reduced, whereas the amount of low-abundance protein was increased, and the sensitivity of mass spectrometry identification was increased. The saturated ammonium sulfate precipitation method may be applied for large-scale urine processing for screening clinical diagnostic markers through proteomics.
Subject(s)
Humans , Histones , Mass Spectrometry , Proteome , Proteomics , UrinalysisABSTRACT
Objective To prepare a wound dressing using nitrocellulose as a membrane and optimize its formulation. Methods Partial analysis was performed on commercial available products. The wound dressings were prepared by using nitrocellulose as a film-forming material, benzyl alcohol as a bacteriostatic agent, castor oil as a plasticizer, isopropyl palmitate as a skin emollient, camphor as a fragrance, and isopropyl alcohol, ethyl acetate and butyl acetate as volatile solvent. The tensile strength, breakpoint elongation percentage, breathability and waterproof performance were tested and evaluated. Results The film-forming performance of the prepared liquid wound dressing was good. The final use amount of nitrocellulose was determined to be 6%. The use amount of plasticizer castor oil was determined to be 4%. Conclusion The prepared liquid wound dressing has good film-forming property, good mechanical property, good waterproof and certain breathability.
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Objetivo: Correlacionar los niveles de anticuerpos antinucleares obtenidos por las técnicas de inmunofluorescencia indirecta (FANA) e inmunoensayo lineal (ANA-LIA) en pacientes diagnosticados o con sospecha clínica de enfermedad autoinmune. Métodos: Se incluyeron 100 pacientes que dieron su consentimiento informado para participar en el estudio. Los ensayos e interpretación de los resultados de las pruebas de FANA y ANA-LIA se realizaron siguiendo las recomendaciones del fabricante. Resultados: el 97,4% de los pacientes estudiados eran de sexo femenino con edad promedio de 42 años, siendo la Artritis reumatoide, Poliartritis y Lupus Eritematoso Sistémico las enfermedades que con mayor frecuencia acompañaron la solicitud médica. Se observó que ambos métodos fueron positivos en el 21% de los casos y que el indice de correlaciòn de Kappa entre las pruebas fue moderado (k= 0,51; p< 0,05), la sensibilidad y especificidad de los métodos fue del 71,4% y 84,8% respectivamente, se observó también que para un determinado patrón fluorescente puede haber positividad de más de un antígeno de la prueba de ANA-LIA y viceversa. Conclusiones: FANA es el método de tamizaje aceptado en la práctica clínica para orientar hacia un diagnóstico clínico de enfermedad autoinmune más probable, debido a la subjetividad en la interpretación de sus resultados y necesidad de entrenamiento en la identificación de los patrones fluorescentes, se requiere del apoyo de otros métodos de laboratorio que permitan identificar con mayor precisión los antígenos reconocidos por los autoanticuerpos, ANA-LIA es una herramienta de laboratorio costo-efectiva de elevada sensibilidad y especificidad que se ajusta a este requerimiento.
Objective: To correlate levels of antinuclear antibodies obtained by indirect immunofluorescence techniques (FANA) and linear immunoassay (ANA-LIA) in patients diagnosed or with clinical suspicion of autoimmune disease. Methods: 100 patients who gave their informed consent to participate in the study were included. Assays and interpretation of results of FANA and ANA-LIA test were performed following the manufacturer's recommendations. Results: 97,4% of the patients studied were female with an average age of 42 years, being rheumatoid arthritis, polyarthritis and systemic lupus erythematosus the diseases that most frequently accompanied the medical request. It was observed that both methods were positive in 21% of the cases and that the Kappa correlation index was moderate between the tests (k = 0,51; p <0,05), the sensitivity and specificity of the methods was 71,4% and 84,8% respectively. It was also observed that for a given fluorescent pattern there may be positivity of more than one antigen of the ANA-LIA test and vice versa. Conclusions: FANA is the screening method accepted in the clinical practice to guide towards a more probable clinical diagnosis of autoimmune disease. Due to the subjectivity in the interpretation of its results and the need for training in the identification of fluorescent patterns, the support of other laboratory methods that allow the identification of antigens recognized by autoantibodies with greater precision is necessary, ANA-LIA is a cost-effective laboratory tool of high sensitivity and specificity that meets this requirement.
Subject(s)
Humans , Antibodies, Antinuclear , Biomarkers , Rheumatic DiseasesABSTRACT
PURPOSE: Nitrocellulose membrane–based filtration system (NCFS) is widely used for protein concentration. In this study, we applied NCFS for production of virus-like particle (VLP) as a vaccine candidate and evaluated yield property and immunogenicity. MATERIALS AND METHODS: Influenza VLPs were generated by baculovirus-insect cell protein expression system. NCFS and sucrose gradient ultracentrifugation were used for purification of VLP. Immunogenicity of VLP was evaluated by animal experiment. RESULTS: Influenza VLPs expressing hemagglutinin (HA) and neuraminidase proteins derived from highly pathogenic influenza virus (H5N8) were effectively produced and purified by NCFS. HA activity of VLP which correlated with antigenicity was well conserved during multiple purification steps. This NCFS based purified VLPs induced influenza virus–specific antibody responses. CONCLUSION: Our results indicate that the influenza VLP vaccine could be prepared by NCFS without loss of immunogenicity and elicit antigen-specific immune responses.
Subject(s)
Animal Experimentation , Antibody Formation , Baculoviridae , Collodion , Filtration , Hemagglutinins , Influenza, Human , Membranes , Neuraminidase , Orthomyxoviridae , Sucrose , Ultracentrifugation , VaccinesABSTRACT
Objective:To solve the inaccurate results caused by the aging of nitrocellulose membranes in qualitative or quantitative immuno-colloidal gold infiltration tests.Methods: Using qualitative TB-DOT and quantitative HCG-DOT immuno-colloidal gold filtration test systems, we compared the treated coloration, circulation velocity, adsorbing power and stability of nitrocellulose membrane of good quality, 3-methyl-1-butanol treated and untreated aged membranes. Results: Abundant results has approved that the regeneration of aged nitrocellulose membrane by 3-methyl-1-butanol treatment lead to 14.4% faster flow velocity, stronger absorption capability, better stability, and lower CV value, compared to untreated membranes.Conclusion:The mentioned method solves the problem caused by the aging of nitrocellulose membranes, and assures the accuracy of measurements. Furthermore, it is an universal solution that can be applied to other POCT products.
ABSTRACT
Introducción: la inmovilización de antígenos a soportes sólidos se utiliza para el desarrollo de diversos inmunoensayos. Una de las primeras tecnologías desarrolladas fue la adsorción de proteínas por aplicación directa sobre la nitrocelulosa. Objetivo: normalizar la inmovilización de un péptido sintético de la proteína de transmembrana gp36 del VIH-2, a un soporte de nitrocelulosa para fines diagnósticos y evaluar los parámetros de desempeño en un grupo de muestras de sueros con reactividad de interés conocida. Métodos: el péptido se inmovilizó de forma libre, conjugado a la albúmina de suero bovina (BSA) y a la hemocianina de lapa marina (KLH) como proteínas portadoras. Se analizaron los parámetros de inmovilización y se determinó la variante óptima. Con la variante escogida se evaluó la sensibilidad y especificidad diagnóstica frente a paneles de referencia del Laboratorio de Investigaciones del SIDA. La especificidad analítica se evaluó con muestras reactivas a VIH-1 y HTLV-I. Resultados: el análisis de las variantes de péptido inmovilizadas a las membranas de nitrocelulosa, demostró que el péptido gp36-BSA, fue el que logró la mayor diferenciación entre muestras positivas y negativas. Se obtuvo 100 por ciento de sensibilidad y 95,2 por ciento de especificidad diagnóstica, así como 100 por ciento de especificidad analítica. Conclusiones: el péptido gp36-BSA inmovilizado en membranas de nitrocelulosa es eficaz en el diagnóstico serológico del VIH-2, lo cual permitirá considerarlo para su empleo con fines diagnósticos en sistemas que utilicen como fase sólida la nitrocelulosa.
Introduction: antigen immobilization in solid supports is used for the development of several immunoassays. One of the first technologies developed was the protein adsorption by direct application to nitrocellulose. Objective: to standardize the immobilization of a synthetic peptide of the HIV-2 transmembrane protein gp36 to nitrocellulose support for diagnostic purposes and to evaluate the performance parameters in a group of serum samples with recognized interesting reactivity. Methods: the peptide was freely immobilized, conjugated to bovine serum albumin (BSA) and to keyhole limpet hemocyanin (KLH) as carrier proteins. Immobilization parameters were analyzed and then, the optimal immobilization alternative was determined. Using the chosen variant, the diagnostic sensitivity and specificity against reference panels of the AIDS Research Laboratory were evaluated. Analytical specificity was evaluated with reactive samples to HIV-1 and HTLV-I. Results: the analysis of the immobilized peptide variants to nitrocellulose membranes showed that the gp36 peptide-BSA was the one that succeeded in setting the greatest differentiation between positive and negative samples. There were observed 100 percent sensitivity, 95.2 percent diagnostic specificity and 100 percent analytical specificity. Conclusions: the gp36-BSA peptide immobilized on nitrocellulose membranes showed efficacy for the serological diagnosis of HIV-2, which will allow considering this peptide for diagnostic uses in systems with nitrocellulose based solid phase.