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ABSTRACT Serodiagnosis of strongyloidiasis is usually performed by ELISA for the detection of IgG antibodies due to its high sensitivity and practicality, but its main limitation is a constant source of S. stercoralis antigens. The use of S. venezuelensis as a heterologous source of antigens has facilitated several published studies on the serodiagnosis and epidemiology of human strongyloidiasis. The main objective of this study was to evaluate the diagnostic accuracy of surface cuticle antigens of infective larvae of S. venezuelensis extracted with CTAB detergent (L3-CTAB) in comparison with soluble somatic extracts (L3-SSE) using a panel of sera from immunocompetent and immunocompromised individuals, at three different cut-offs. ROC curve analysis showed that L3-CTAB had an AUC of 0.9926. At the first cut-off value (OD 450 nm = 0.214), sensitivity and specificity were 100% and 90.11%, respectively, with a diagnostic accuracy of 0.93. At a second cut-off value (OD 450 nm = 0.286), sensitivity and specificity were 70% and 100%, respectively, with a diagnostic accuracy of 0.91. However, at an alternative third cut-off value (OD 450 nm = 0.589), sensitivity and specificity were 95% and 97.8%, respectively, with a diagnostic accuracy of 0.97. Using L3-CTAB as an antigenic source, the seropositivity rate in immunocompromised patients was 28.13% (9/32) whereas a seropositivity rate of 34.38% (11/32) was found when L3-SSE was used in ELISA. Therefore, the L3-CTAB is simple and practical to obtain and was found to be highly sensitive and specific.
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ABSTRACT Introduction: Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Patients with SLE exhibit multiple serum autoantibodies, including anti-neutrophil cytoplasmic antibodies (ANCAs). There are two main techniques to detect ANCAs: indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA). In this study, an attempt was made to determine the frequency and clinical associations of ANCAs in patients with SLE. Methods: A cross-sectional study was conducted in a tertiary care hospital in Colombia that included 74 patients with SLE. The presence of ANCAs was assessed using IIF with ethanol-fixed slides, and ELISA was used to detect antibody specificities for myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA. Results: Of the 74 patients with SLE evaluated, 60 (81.1%) of them were ANCA-positive by IIF. By contrast, only one patient showed specificity for PR3-ANCA by ELISA. The relevance of ANCA positivity by IIF and clinical and serological features was significant for renal involvement (p = .0174), and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (p = .0308). Conclusion: ANCAs are common in the serum of patients with SLE, as detected by ethanol-fixed slides with IIF staining. However, detection of specificity to PR3 and/or MPO is rare, thus highlighting the importance of detecting these autoantibodies by different techniques.
RESUMEN Introducción: El lupus eritematoso sistémico (LES) es una enfermedad autoinmune sistémica. Los pacientes con LES muestran múltiples autoanticuerpos séricos, incluyendo los anticuerpos anticitoplasma de neutrófilo (ANCA, por sus siglas en inglés). Existen 2 técnicas principales para la detección de ANCA: inmunofluorescencia indirecta (IFI) y ensayo por inmunoadsorción ligado a enzimas (ELISA). En este estudio nuestro objetivo fue determinar la frecuencia y las asociaciones clínicas de los ANCA en pacientes con LES. Métodos: Realizamos un estudio transversal de 74 pacientes con LES en un hospital de alta complejidad de Colombia. La presencia de ANCA se evaluó por IFI, utilizando láminas con fijación de etanol, y con ELISA para determinar las especificidades para mieloperoxidasa (MPO)-ANCA y proteinasa 3 (PR3)-ANCA. Resultados: Fueron evaluados 74 pacientes con LES, 60 (81,1%) de ellos fueron positivos para ANCA. Por el contrario, solo un paciente mostró especificidad para PR3-ANCA por ELISA. La relación entre la positividad para ANCA por IFI y las características clínicas y serológicas fue estadísticamente significativa para compromiso renal (p = 0,0174) y para el índice de actividad de la enfermedad (Systemic Lupus Erythematosus Disease Activity Index [SLEDAI]) (p = 0,0308). Conclusiones: Los ANCA detectados mediante fijación con etanol por técnicas de IFI, son comunes en pacientes con LES. Sin embargo, la detección de especificidades para PR3 o MPO es rara; se destaca la importancia de la evaluación de estos autoanticuerpos mediante diferentes técnicas.
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INTRODUCCIÓN: La enfermedad de Chagas es una infección parasitaria crónica sistémica, de importancia global, causada por Trypanosoma cruzi. OBJETIVO: Determinar la prevalencia de anticuerpos contra T cruzi en mujeres embarazadas en el estado de Morelos, México. MATERIALES Y MÉTODOS: Se analizaron 1.620 sueros de mujeres embarazadas mediante dos pruebas serológicas: ELISAc (antígeno crudo nativo) y ELISAr (antígeno recombinante, no nativo). Las muestras reactivas se analizaron posteriormente mediante hemaglutinación indirecta (HAI). Se utilizaron dos enfoques de detección, en paralelo (son positivas las muestras reactivas por cualquier método) y en serie (son positivas las muestras confirmadas por HAI). Se evaluaron factores sociodemográficos y de salud asociados a la presencia de anticuerpos contra T. cruzi mediante razones de momios al 95%. RESULTADOS: Se obtuvo una seroprevalencia de 4,87% con el diagnóstico en paralelo y de 0,43% en serie. A partir de los resultados en paralelo las mujeres que fueron atendidas en los hospitales generales de Tetecala y Jojutla tuvieron, respectivamente, 2,2 y 2,0 veces mayor posibilidad de presentar anticuerpos contra T cruzi con respecto a las mujeres que fueron atendidas en el Hospital General de Cuautla. CONCLUSIÓN: La prevalencia de anticuerpos contra T cruzi en mujeres embarazadas en el estado de Morelos fluctuó entre 0,43 y 4,87%, según el antígeno y el abordaje utilizado. Es necesario continuar con la vigilancia de la seroprevalencia de anticuerpos contra T cruzi en mujeres embarazadas en el estado de Morelos, México, con las técnicas de mayor sensibilidad y especificidad disponibles.
BACKGROUND: Chagas disease is a globally important chronic systemic parasitic infection caused by Trypanosoma cruzi. AIM: To determine the prevalence of antibodies against T cruzi in pregnant women from the state of Morelos, México. METHODS: 1,620 sera from pregnant women were analyzed using two serological tests: ELISAc (native crude antigen) and ELISAr (recombinant, non-native antigen). Reactive samples were subsequently analyzed by indirect hemagglutination (IHA). Two detection approaches were used, in parallel (reactive samples by any method are positive) and serial (samples confirmed by IHA are positive). Sociodemographic and health factors associated with the presence of antibodies against T cruzi were evaluated using 95% odds ratios. RESULTS: A seroprevalence of 4.87% was obtained with parallel diagnosis and 0.43% in series. From the parallel results, the women who were attended at the general hospitals of Tetecala and Jojutla had respectively 2.2 and 2.0 times greater chance of presenting antibodies against T cruzi compared to the women who were attended at the General Hospital of Cuautla. CONCLUSION: The prevalence of antibodies against T cruzi in pregnant women from the state of Morelos fluctuated between 0.43 and 4.87%, depending on the antigen and the approach used. It is necessary to continue with the surveillance of the seroprevalence of antibodies against T cruzi in pregnant women from the state of Morelos, Mexico, using the techniques with the highest sensitivity and specificity available.
Subject(s)
Humans , Female , Pregnancy , Trypanosoma cruzi , Chagas Disease/diagnosis , Chagas Disease/epidemiology , Enzyme-Linked Immunosorbent Assay/methods , Antibodies, Protozoan , Seroepidemiologic Studies , Pregnant Women , Mexico/epidemiologyABSTRACT
Objective:To evaluate the value of indirect immunofluorescence on salt-split skin (IIF-SSS) in the diagnosis of bullous pemphigoid (BP) .Methods:A single-center clinical retrospective study was conducted. Totally, 163 patients with newly diagnosed BP were collected from Hospital of Dermatology, Chinese Academy of Medical Sciences from January 2013 to January 2019, so were 404 controls, including 161 with pemphigus, 67 with eczema, 26 with drug eruption, 23 with erythema multiforme, 18 with prurigo nodularis, etc. Blood samples were collected before the treatment, and IIF-SSS, BP180 NC16A enzyme-linked immunosorbent assay (ELISA) and direct immunofluorescence (DIF) assay were performed to evaluate the value of IIF-SSS in the diagnosis of BP. Measurement data were compared by using t test and Mann-Whitney test, and enumeration data were compared by using chi-square test and Fisher′s exact test or McNemar test. Results:The number of cases positive for IIF-SSS, BP180 NC16A ELISA and DIF assay was 160, 153 and 127 respectively in the BP group, and 0, 18 and 26 respectively in the control group. The sensitivities of IIF-SSS, BP180 NC16A ELISA and DIF assay for the diagnosis of BP were 98.15%, 93.86% and 77.91% respectively, and their specificities were 100%, 95.54% and 93.56% respectively. There was strong consistency in the diagnosis of BP between IIF-SSS and DIF (Kappa coefficient= 0.767, P < 0.001) . Conclusion:IIF-SSS has relatively high sensitivity and specificity for the diagnosis of BP, and can serve as a routine method for diagnosing BP.
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ObjectiveTo compare the effect of different solvent extracts of spore powder and fruiting body of Lasiosphaera Calvatia on fibroblasts and wound healing of full-thickness skin defect， in order to screen the optimal medication part of Lasiosphaera Calvatia. MethodThe effect of water extract and alcohol extract of spore powder and fruiting body on cell proliferation and cell migration of mouse skin fibroblasts （MSF） were examined in vitro. Cell proliferation and activity test （CCK-8） method was used for cell proliferation， scratch assay was used for cell migration， flow cytometry was conducted to explore cell cycle， enzyme-linked immunosorbent assay （ELISA） was used to determine the production of collagen Ⅰ and Ⅲ. At the same time， a full-thickness skin defect wound model was established to investigate the therapeutic effect of different solvent extracts of spore powder. Ultraviolet-visible spectrophotometry was used to measure the contents of index components in different solvent extracts. ResultThe water extract of spore powder and fruiting body had certain cytotoxicity， while the alcohol extract could promote proliferation， migration and production of collagen Ⅰ and Ⅲ of MSF， and the effect of spore powder was significantly higher than that of fruiting body. When the concentration was 10 mg·L-1， the cell proliferation rate of alcohol extract of spore powder was as high as （159.22±15.95）%， and could promote MSF from the G0/G1 phase to S phase and G2/M phase with an increased proliferation index. The alcohol extract also promoted the migration of fibroblasts， secreted collagen Ⅰ and Ⅲ. On in vivo model， the alcohol extract of spore powder significantly accelerated wound healing on mice， effectively promoted the complete epithelialization of wound tissue， and generated new collagen fiber. The results of determination showed that the contents of polyphenols and flavonoids in the alcohol extract were higher than the alcohol extract of fruiting body. ConclusionThe alcohol extract of spore powder in Lasiosphaera Calvatia has active components in the treatment of wounds with good development prospect， and the medicinal components may be polyphenols and flavonoids.
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Objective To evaluate the diagnostic efficiency of four anti-cysticercus IgG, IgG4 or IgM antibody test kits (enzyme-linked immunosorbent assay, ELISA) by different manufacturers, so as to provide insights into the epidemiological investigation and clinical detection of cysticercosis. Methods Forty serum samples from cerebral cysticercosis patients, 100 serum samples from healthy volunteers, 30 serum samples from paragonimiasis skrjabini patients, 17 serum samples from cystic echinococcosis and 19 serum samples from subcutaneous or cerebral sparganosis patients were collected and detected using anti-cysticercus IgG, IgG4 or IgM antibody test kits (brand A) and the anti-cysticercus IgG antibody test kit (brand B). The sensitivity, specificity and false negative rate of the four kits for detection of cysticercosis were estimated. Results The anti-cysticercus IgG, IgG4 or IgM antibody test kits (brand A) showed 95.00% (38/40), 87.50% (35/40), 7.50% (3/40) sensitivities and 98.00% (98/100), 100.00% (100/100) and 100.00% (100/100) for detection of cysticercosis, while the anti-cysticercus IgG antibody test kit (brand B) presented a 75.00% (30/40) sensitivity and 100.00% (100/100) specificity for detection of cysticercosis. The sensitivity for detection of cysticercosis was significantly higher by the anti-cysticercus IgG antibody test kit (brand A) than by the anti-cysticercus IgG antibody test kit (brand B) (χ2 = 6.28, P < 0.05); however, no significant difference was seen in the specificity by two kits (χ2 = 2.01, P > 0.05). The four ELISA kits showed overall false positive rates of 37.88% (25/66), 22.73% (15/66), 62.12% (41/66) and 15.15% (10/66) for detection of paragonimiasis, echinococcosis and sparganosis (χ2 = 37.61, P < 0.05), and the anti-cysticercus IgG antibody test kit (brand A) presented the highest overall false positive rate for detection of paragonimiasis, echinococcosis and sparganosis (χ2 = 7.56, P’ < 0.008), while a higher overall false positive rate was seen for detection of paragonimiasis, echinococcosis and sparganosis by the anti-cysticercus IgG antibody test kit (brand A) than by the anti-cysticercus IgG antibody test kit (brand B) (χ2 = 8.75, P’ < 0.008). The four ELISA kits showed false positive rates of 40.00% (12/30), 16.67% (5/30), 76.67% (23/30) and 13.33% (4/30) for detection of paragonimiasis (χ2 = 32.88, P < 0.05) and 21.05% (4/19), 26.32% (5/19), 73.68% (14/19) and 15.79% (3/19) for detection of sparganosis (χ2 = 19.97, P < 0.05), and the highest false positive rates were found by the anti-cysticercus IgM antibody test kit (brand A) for detection of paragonimiasis and sparganosis (all P’ < 0.008). However, the four ELISA kits showed comparable false positive rates of 52.94% (9/17), 29.41% (5/17), 23.53% (4/17) and 17.65% (3/17) for detection of echinococcosis (χ2 = 8.24, P > 0.05). In addition, the anti-cysticercus IgM anti-body test kit (brand A) showed false positive rates of 76.67% (23/30), 23.53% (4/17) and 73.68% (14/19) for detection of paragonimiasis, echinococcosis and sparganosis (χ2 = 14.537, P < 0.05), with the lowest false positive rate seen for detection of echinococcosis (χ2 = 14.537, P’ < 0.014), while no significant differences were seen in the false positive rate for detection of paragonimiasis, echinococcosis and sparganosis by other three ELISA kits (all P > 0.05). Conclusions The four anti-cysticercus IgG, IgG4 or IgM antibody test kits exhibit various efficiencies for serodiagnosis of cysticercosis. The anti-cysticercus IgG antibody test kit (brand A) has a high sensitivity for serodiagnosis of cysticercosis; however, it still needs to solve the problems of cross-reaction with other parasitic diseases and stability.
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Puerarin was conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) by periodate oxidation to serve as the immunogen and coating antigen, respectively. BALB/c mice were immunized with puerarin-BSA according to the routine immunization procedure, and the titer and specificity of serum were detected after three immunization. After booster immunization, mouse spleen lymphocytes were fused with mouse myeloma cells, and 24 hybridoma cell lines of the monoclonal antibodies against puerarin were screened by monoclonal antibody screening technique. Ascites was prepared and purified. The cross-reactivity of monoclonal antibody(mAb) M1 with 4'-methoxy puerarin, daidzin, puerarin-6″-O-xyloside, daidzein, mirificin, 3'-methoxy puerarin, and 3'-hydroxy puerarin was 239.84%, 112.18%, 67.89%, 58.28%, 22.37%, 0.40%, and 0.20%, respectively, and those with other analogs such as baicalein and baicalin were all less than 0.10%. The IC_(50) and the working range of the indirect competitive enzyme-linked immunosorbent assay(icELISA) for puerarin were 44.80 ng·mL~(-1) and 8.20-292.30 ng·mL~(-1), respectively. The average recovery was 91.95%-98.20% with an RSD in the range of 0.70%-2.60%. The content of puerarin in different Puerariae Lobatae Radix samples was determined with icELISA and validated by UPLC-MS. The correlation between data obtained from icELISA and UPLC-MS was 0.999 0, indicating that icELISA is suitable for the rapid detection of puerarin in Puerariae Lobatae Radix samples.
Subject(s)
Animals , Antibodies, Monoclonal , Chromatography, Liquid , Enzyme-Linked Immunosorbent Assay/methods , Hybridomas/metabolism , Isoflavones , Mice , Mice, Inbred BALB C , Tandem Mass SpectrometryABSTRACT
Foot and mouth disease is a highly contagious viral disease of cloven-hoofed animals that has a significant economic impact on livestock. A recent outbreak was detected and recorded as exotic strain of foot and mouth disease virus SAT2 (Serotype SAT2, topotype VII, Lib-12 lineage). The emergency vaccine was produced and assessed in vivo and large number of vaccine batches were urgently needed. The present work was aimed to provide a rapid evaluation of inactivated foot and mouth disease SAT2 oily vaccine to exclude the unsatisfactory batches during emergency circumstances and to reduce time, effort and cost. The extraction of foot and mouth disease antigen content from oily adjuvanted vaccine was carried out using isopropyl myristate and benzyl alcohol methods. The extracted viral antigen was identified by foot and mouse disease serotyping ELISA and 146S content was quantified using sucrose density gradient analysis. Evaluations were carried out instantly and at 2h, 6h and 24h. The results indicated the efficiency of benzyl alcohol to breakdown the oil emulsion either MONTANIDE™ ISA 206 VG or MONTANIDE™ ISA 50 V2, while the isopropyl myristate was efficient for MONTANIDE™ ISA 50 V2 only. The identification and quantification of 146S for extracted antigen using benzyl alcohol indicated significant stable records at different time intervals for the vaccine batches, while the extraction using isopropyl myristate indicated unstable records at different time intervals. It was concluded that the evaluation of monovalent foot and mouse disease vaccine could be conducted in vitro, using serotyping ELISA and quantification of 146S for the extracted antigen, either using benzyl alcohol or isopropyl myristate (MONTANIDE™ ISA50 V2 only), with the consideration that 146S content should not less than 4 μg/mL(AU)
La fiebre aftosa es una enfermedad viral altamente contagiosa de los animales de pezuña hendida que tiene un impacto económico significativo en el ganado. Se detectó un brote reciente que se registró como causado por una cepa exótica del virus de la fiebre aftosa (serotipo SAT2, topotipo VII, linaje Lib-12). La vacuna de emergencia se elaboró y evaluó in vivo, existiendo una urgente necesidad de contar con un gran número de lotes de la misma. El presente trabajo tuvo como objetivo proporcionar una evaluación rápida de la vacuna oleosa inactivada (SAT2) contra la fiebre aftosa, para excluir los lotes insatisfactorios durante circunstancias de emergencia, reduciendo tiempo, esfuerzo y costo. La extracción del contenido de antígeno de fiebre aftosa, de la vacuna oleosa adyuvada, se llevó a cabo utilizando miristato de isopropilo y alcohol bencílico. El antígeno viral extraído se identificó utilizando un ELISA de serotipificación y se cuantificó el contenido de 146S mediante análisis de gradiente de densidad de sacarosa. Las evaluaciones se realizaron de forma instantánea y a las 2h, 6h y 24h. Los resultados indicaron la eficacia del alcohol bencílico para separar la emulsión de aceite para MONTANIDE ™ ISA 206 VG o MONTANIDE™ ISA 50 V2, mientras que el miristato de isopropilo fue eficaz para MONTANIDE™ ISA 50 V2 únicamente(AU)
Subject(s)
Animals , Enzyme-Linked Immunosorbent Assay/methods , Foot-and-Mouth Disease , Vaccines , EgyptABSTRACT
Abstract: Dogs are the main reservoirs in the domestic transmission cycle of visceral leishmaniasis, and the diagnosis is essential for the effectiveness of the control measures recommended by the Brazilian Ministry of Health. We assessed the diagnostic performance of the ELISA-Vetlisa/BIOCLIN prototype with serum samples from 200 dogs, in triplicate, including symptomatic, oligosymptomatic, asymptomatic, and healthy dogs, originated by two distinct panels (A and B) characterized by parasitological tests as the reference standard. In this study, the prototype kit showed a 99% sensitivity (95%CI: 94.5-100.0) and a 100% specificity (95%CI: 96.4-100.0). The sensitivity of the prototype kit did not vary significantly with the clinical status of the dogs. Considering the final result classification (positive or negative), agreement between the results of repeated tests was almost perfect (kappa = 0.99; 95%CI: 0.98-1.00). ELISA-Vetlisa/BIOCLIN is a promising option for the serological diagnosis of canine visceral leishmaniasis in Brazil.
Resumo: Os cães são os principais reservatórios do ciclo de transmissão domiciliar da leishmaniose visceral, e o diagnóstico é essencial para a efetividade das medidas de controle recomendadas pelo Ministério da Saúde. Os autores avaliam o desempenho diagnóstico do protótipo da ELISA-Vetlisa/BIOCLIN em amostras sorológicas de 200 cães, em triplicata, incluindo cães sintomáticos, oligossintomáticos e saudáveis, com dois painéis distintos (A e B) caracterizados por testes parasitológicos enquanto referência. No estudo, o kit-protótipo mostrou sensibilidade de 99% (IC95%: 94,5-100,0) e especificidade de 100% (IC95%: 96,4-100,0). A sensibilidade do kit-protótipo não variou de maneira significativa de acordo com o estado clínico dos cães. Considerando a classificação final dos resultados (positivo ou negativo), a concordância entre os resultados dos testes em triplicata foi quase perfeita (kappa = 0,99; IC95%: 0,98-1,00). O protótipo ELISA-Vetlisa/BIOCLIN tem o potencial de ser utilizada para o diagnóstico sorológico da leishmaniose visceral canina no Brasil.
Resumen: Los perros son los principales reservorios en el ciclo de transmisión doméstica de la leishmaniasis visceral, por ello el diagnóstico es esencial para la efectividad de las medidas de control recomendadas por el Ministerio de Salud de Brasil. Evaluamos el desempeño diagnóstico de ELISA-Vetlisa/BIOCLIN prototipo con muestras de sérum de 200 perros, en triplicado, incluyendo sintomático, oligosintomático, asintomático y perros sanos, originadas por dos paneles distintos (A y B), caracterizados por test parasitológicos como referencia estándar. En este estudio, el kit prototipo mostró un 99% de sensibilidad (IC95%: 94,5-100,0) y un 100% de especificidad (IC95%: 96,4-100,0). La sensibilidad del kit prototipo no varió significativamente con el estatus clínico de los perros. Considerando la clasificación final del resultado (positiva o negativa), el acuerdo entre los resultados de los tests repetidos fue casi perfecto (kappa = 0,99; IC95%: 0,98-1,00). ELISA-Vetlisa/BIOCLIN tiene potencial para ser usado para el diagnóstico serológico de la leishmaniasis visceral canina en Brasil.
Subject(s)
Animals , Dogs , Leishmania infantum , Dog Diseases/diagnosis , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/veterinary , Brazil , Enzyme-Linked Immunosorbent Assay , Sensitivity and SpecificityABSTRACT
Objective:To establish a safe, effective, simple, and more economically feasible method to obtain platelet-rich plasma (PRP).Methods:Whole blood was collected from 24 patients with atrophic acne scars on the face. For the preparation of PRP, a slight modification of Choukroun＇s method was used. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of platelet derived growth factor-β(PDGF-β), transforming growth factor-β1 (TGF-β1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), isulin-like growth factor-1 (IGF-1) in normal plasma and PRP.Results:The average concentration of PDGF-β in PRP was (755.61±418.31) ng/L, which was higher than [(479.93±279.18) ng/L] in normal plasma ( t=3.479, P<0.01). The average concentration of TGF-β1 in PRP was (2267.00±1223.68), which was higher than [(1535.50±910.91) ng/L] in normal plasma ( t=7.082, P<0.01). The average concentration of EGF in PRP was (30.70±12.39) ng/L, which was higher than [(20.77±10.31) ng/L] in normal plasma ( t=6.899, P<0.01). The average concentration of VEGF in PRP was (25.42±17.69) ng/L, which was higher than [(12.01±7.77) ng/L] in normal plasma ( t=5.230, P<0.01). The average concentration of bFGF was (17.85±7.17) ng/L, which was higher than [(10.90±4.73) ng/L] in normal plasma ( t=6.050, P<0.01). The average concentration of IGF-1 was (201.22±36.80) ng/ml, which was higher than [(174.90±33.80) ng/ml] in normal plasma ( t=3.760, P<0.01). Conclusions:Compared to normal plasma, modified PRP contains higher levels of growth factors. The modified method is a reliable option for liquid PRP, especially applicable for invasive cosmetic laser surgery to promote wound repair.
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The indirect competitive enzyme-linked immunosorbent assay(ic-ELISA) and direct competitive enzyme-linked immunosorbent assay(dc-ELISA) were performed for the rapid detection of aflatoxin B_1(AFB_1) in Astragali Radix, Zingiberis Rhizoma Recens, Jujubae Fructus, and Nelumbinis Semen with self-made antigens and antibodies. Different extraction methods were investigated to reduce the matrix effects of different medicinal parts in Chinese herbal medicines. The sensitivity of dc-ELISA method was improved by optimizing the molar ratio of AFB_1 to horseradish peroxidase(HRP). In this study, the sensitivity(IC_(50)) of ic-ELISA and dc-ELISA was 0.046 and 0.023 ng·mL~(-1), with the limit of detection(LOD) of 0.007 and 0.004 ng·mL~(-1), respectively. The detection time was 3 h and 50 min for ic-ELISA and dc-ELISA, respectively. The recovery rates were within the range of 62.96%-104.4%, with RSDs of less than 10%. Confirmed by LC-MS/MS, three positive samples of Nelumbinis Semen were detected from 53 samples. Two ELISA methods established in this study were accurate, rapid and sensitive, and can be used for rapid screening of AFB_1 in Chinese herbal medicines such as Astragali Radix, Zingiberis Rhizoma Recens, Jujubae Fructus, and Nelumbinis Semen. In addition, the advantages and limitations of the two methods were compared and discussed, which can provide a reference for the testing institutions to choose the proper method.
Subject(s)
Aflatoxin B1/analysis , China , Chromatography, Liquid , Enzyme-Linked Immunosorbent Assay , Tandem Mass SpectrometryABSTRACT
Objective:To explore effects of different extracts and monomers of <italic>Lepidium meyenii </italic>（Maca） on the proliferation of mouse splenic lymphocytes and induction of interleukin-2 （IL-2） and tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） by observing their immunomodulatory effects. Method:An octadecylsilyl （ODS） column was used to enrich the methanol extract of <italic>L. meyenii</italic> in stages to obtain six fractions and three monomers. Different groups of extracts and monomers of <italic>L. meyenii </italic>at different doses were set up. Cell counting Kit-8 （CCK-8） was used to detect the effect on the proliferation of mitogen-free， concanavalin A （Con A）-induced， and lipopolysaccharides （LPS）-induced mouse splenic lymphocytes. Enzyme-linked immunosorbent assay （ELISA） was used to determine the levels of IL-2 and TNF-<italic>α</italic>. Result:<italic>L. meyenii </italic>extracts Fr<sub>3</sub> and Fr<sub>6</sub>， and monomers <italic>N</italic>-benzyl hexadecanamide and 1，2-dihydro-4-carboxaldehyde-3-benzyl-<italic>N</italic>-hydroxypyridine slightly promoted the proliferation of Con A-induced T lymphocytes and LPS-induced B lymphocytes （<italic>P</italic><0.01） as compared with the conditions in the model group. <italic>L. meyenii</italic> extracts and monomers significantly induced the secretion of IL-2 and TNF-<italic>α</italic> by splenic lymphocytes （<italic>P</italic><0.01）. Conclusion:<italic>L. meyenii</italic> extracts and monomers can achieve immunological enhancement by promoting the secretion of IL-2 and TNF-<italic>α</italic>， and facilitate the proliferation of splenic lymphocytes. The active components are presumedly macamides and pyridine alkaloids， and the specific mechanism still needs to be further explored.
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Objective:To explore a fast method to identify and confirm suspected clonorchis sinensis infection.Methods:For suspected clonorchis sinensis infection, the clonorchiasis serum antibody was detected first with ELISA. If the antibody was positive, the fecal examination for eggs was performed. If the fecal examination was negative, duodenal drainage under gastroscopy was recommended to detect eggs from the drainage fluid.Results:A total of 126 patients met the requirements and aged 54.14±13.33 (24- 87). There were 83 cases (65.87%, 83/126) with eggs positive in the drainage fluid, of which 53 cases were male, aged 55.91±11.47 (30-86), and 30 cases female, aged 55.87± 13.85(30-87). There was no significant difference in age between males and females( P>0.05). The time of catheterization (T1) of 126 cases was 3.79 ±1.45 min. The time of drainage (T2) of 126 cases was 31.39 ±14.29 min. There was no significant difference in T1 or T2 between the positive group and the negative group( P>0.05). The detection rates of eggs were 91.57% (76 cases) in intrahepatic bile duct drainage, 81.93% (68 cases) in the bile-cyst juice and 75.90% (63 cases) in the common bile duct fluid. No serious adverse reactions occurred during or after the operation. Conclusion:The detection rate of clonorchiosis sinensis can be effectively improved by the combination of clonorchiasis serum antibody test and gastroscopy-guided duodenal drainage.
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Objective:To detect IgG and neutralizing antibodies response to SARS-CoV-2 vaccine by comparing enzyme-linked immunosorbent assay (ELISA), commercial magnetic particle chemiluminescence assay(CLIA) and neutralization test(NT).Methods:ELISA, CLIA and NT were used to detect 143 healthy people before and after 28 days immunization with 2 doses of SARS-CoV-2 vaccine, and calculate the positive conversion rate, quantitative results and analysis the consistency of the three methods.Results:The positive conversion rate of SARS-CoV-2 vaccine antibody detected by ELISA, CLIA and NT were respectively 97.9%, 98.6% and 85.3%. The geometric mean of the highest dilution of the serum quantitatively detected by ELISA was 586.6; The mean of CLIA S/CO value was 11.26; The geometric mean titer of the NT was 7.6. The correlation coefficient between ELISA, CLIA and NT were respectively 0.69( P<0.01) and 0.65( P<0.01), and the correlation coefficient between ELISA and CLIA was 0.79( P<0.01). Conclusions:The three methods all detected high levels of antibodies response to SARS-CoV-2 vaccine immunization. ELISA and CLIA are more consistent to detect IgG antibody, and have a good correlation with the quantitative detection results of the NT.
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Objective:To compare the results of three detection methods, single antigen-bead assay(SAB), Luminex screening assay(LMX), and ELISA assay for detecting HLA antibody, and compares the two screening methods, LMX and ELISA with SAB detection as a reference method to provide a reference for organ transplantation laboratories to choose a reasonable HLA antibody test strategy.Methods:A lot of 124 consecutive samples were tested using SAB, ELISA, and LMX methods at the same time, and analyze the differences of these results. SAB testing was used as a reference method to analyze the sensitivity and specificity of the two screening assays. Chi-square analysis was used to compare the two methods, and P<0.05 was considered statistically significant. Results:Both ELISA and LMX methods showed low sensitivity of 34.4% and 31.3% for HLA class I, and 29.7% and 51.3% for class Ⅱ. Otherwise, the specificity of the ELISA and LMX method was much higher. For class, I both was 98.9%, and for class Ⅱ were 100% and 91.9% respectively. Out of 124 samples, the number of SAB(+ )ELISA(-)LMX(-) results was 17, and SAB(-)ELISA(+ )LMX(+ ) results was zero indicating that there were considerably screening assays probably with missed detection. In the cases of SAB(+ )ELISA(-)LMX(-), the distribution of MFI value of SAB assay ranges from 750 to 7000.Conclusions:Because the sensitivity of the two screening methods is relatively low, there is a greater risk of missed antibody detection in the scheme of testing for specific antibodies after the screening test is positive. This should be paid attention to, especially for patients with a history of sensitization. For negative screening test results, SAB or other assays should be considered to check the result. It could provide more accurate results when SAB which is recognized as higher sensitivity and specificity is directly used as an initial test. At the same time, the MFI value of the SAB test can serve as an indicator to determine whether to add other assays to check the ASB result.
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In canonical Wnt/β-catenin signaling pathway, β-catenin/TCF4 (T-cell factor 4) interaction plays an important role in the pathogenesis and development of non-small cell lung cancer (NSCLC), and it is tightly associated with the proliferation, chemoresistance, recurrence and metastasis of NSCLC. Therefore, suppressing β-catenin/TCF4 interaction in Wnt/β-catenin signaling pathway would be a new therapeutic avenue against NSCLC metastasis. In this study, considering the principle of enzyme-linked immunosorbent assay (ELISA), an optimized high-throughput screening (HTS) assay was developed for the discovery of β-catenin/TCF4 interaction antagonists. Subsequently, this ELISA-like screening assay was performed using 2 μg/mL GST-TCF4 βBD and 0.5 μg/mL β-catenin, then a high Z' factor of 0.83 was achieved. A pilot screening of a natural product library using this ELISA-like screening assay identified plumbagin as a potential β-catenin/TCF4 interaction antagonist. Plumbagin remarkably inhibited the proliferation of A549, H1299, MCF7 and SW480 cell lines. More importantly, plumbagin significantly suppressed the β-catenin-responsive transcription in TOPFlash assay. In short, this newly developed ELISA-like screening assay will be vital for the rapid screening of novel Wnt inhibitors targeting β-catenin/TCF4 interaction, and this interaction is a potential anticancer target of plumbagin in vitro.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , High-Throughput Screening Assays , Humans , Lung Neoplasms , Transcription Factor 4/genetics , beta Catenin/geneticsABSTRACT
@#Among current novel druggable targets, protein–protein interactions (PPIs) are of considerable and growing interest. Diacylglycerol kinase α (DGKα) interacts with focal adhesion kinase (FAK) band 4.1-ezrin-radixin-moesin (FERM) domain to induce the phosphorylation of FAK Tyr397 site and promotes the malignant progression of esophageal squamous cell carcinoma (ESCC) cells. Chrysin is a multi-functional bioactive flavonoid, and possesses potential anticancer activity, whereas little is known about the anticancer activity and exact molecular mechanisms of chrysin in ESCC treatment. In this study, we found that chrysin significantly disrupted the DGKα/FAK signalosome to inhibit FAK-controlled signaling pathways and the malignant progression of ESCC cells both in vitro and in vivo, whereas produced no toxicity to the normal cells. Molecular validation specifically demonstrated that Asp435 site in the catalytic domain of DGKα contributed to chrysin-mediated inhibition of the assembly of DGKα/FAK complex. This study has illustrated DGKα/FAK complex as a target of chrysin for the first time, and provided a direction for the development of natural products-derived PPIs inhibitors in tumor treatment.
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RESUMEN La Paratuberculosis Bovina (PTB) o Enfermedad de Johne, es una infección del tracto gastrointestinal causada por Mycobacterium avium, subespecie paratuberculosis (Map), que se caracteriza por generar enteritis granulomatosa crónica y linfadenitis en rumiantes. La preocupación más relevante en relación con la importancia de la PTB es su posible vínculo con la Enfermedad de Crohn (EC) en humanos, sin embargo, esta asociación aún está bajo investigación. Se determinó la seroprevalencia de PTB en el municipio de Sogamoso (Boyacá), donde se recolectaron 604 muestras de sangre, cuyo suero fue procesado mediante la técnica de ELISA indirecta con el kit comercial PARACHEK® 2 KIT (Prionics, Suiza). La seroprevalencia fue de 10,9% (66/604), donde el grupo etario de 2 a 3 años y la raza Jersey fueron los de mayor seroprevalencia. Se encontró asociación estadística significativa (p≤0,05) entre la edad de los individuos evaluados y el suministro de concentrado. La seroprevalencia encontrada indica que se está produciendo una transmisión activa de la enfermedad y que las medidas de control disponibles no están siendo llevadas a cabo o no son lo suficientemente efectivas.
ABSTRACT Bovine Paratuberculosis (BPT), or Johne's Disease, is an infection of the gastrointestinal tract caused by Mycobacterium avium, subspecies paratuberculosis (Map), which is characterized by chronic granulomatous enteritis and lymphadenitis in ruminants. The most relevant concern regarding the importance of BPT is its possible link to Crohn's disease (CD) in humans, however this association is still under investigation. The seroprevalence of BPT was determined in the municipality of Sogamoso (Boyacá), where 604 blood samples were collected, their serum was processed by the indirect ELISA technique with the commercial PARACHEK® 2 KIT (Prionics, Switzerland), following the manufacturer's instructions. The seroprevalence was 10,9% (66/604), with the 2 to 3 years age group and the Jersey breed having the highest seroprevalence. A significant statistical association was found (p≤0,05) with the age of the individuals tested and the supply of concentrate. The seroprevalence found indicates that active transmission of the disease is taking place, and that the available control measures are not being carried out or are not effective enough.
RESUMO A Paratuberculose Bovina (PTB) ou Doença de Johne é uma infecção do trato gastrointestinal causada pela Mycobacterium avium subespécie paratuberculosis (Map), caracterizada por gerar enterite granulomatosa crónica e linfadenite em ruminantes. A preocupação mais relevante em relação à importância do PTB é seu possível vínculo com a Doença de Crohn (DC) em seres humanos, no entanto, essa associação ainda está sob investigação. A soroprevalência do PTB foi determinada no município de Sogamoso (Boyacá), onde foram coletadas 604 amostras de sangue, cujo soro foi processado pela técnica ELISA indireta com o kit comercial PARACHEK® 2 KIT (Prionics, Suíça). A soroprevalência foi de 10,9% (66/604), onde a faixa etária de 2 a 3 anos e a raça Jersey foram as que apresentaram maior soroprevalência. Foi encontrada associação estatisticamente significante (p≤0,05) com a idade dos indivíduos avaliados e o suprimento de concentrado. A soroprevalência encontrada indica que a transmissão ativa da doença está ocorrendo e que as medidas de controle disponíveis não estão sendo realizadas ou não são eficazes o suficiente.
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Enzootic bovine leukosis (EBL) is an infectious disease caused by bovine leukemia virus (BLV) that affects cattle worldwide. Agar gel immunodiffusion (AGID) was the reference test for EBL diagnosis for many years, but enzyme-linked immunosorbent assay (ELISA) showed higher sensitivity, was faster to perform, and resulted in an objective reading. However, the importation of ELISA kits is lengthy and expensive, and currently, no AGID kits are available in Brazil. The aim of this work was to standardize an indirect ELISA (iELISA) for EBL diagnosis using BLV antigens produced in Tadarida brasiliensis lung (Tb1Lu) cells, which are Bovine viral diarrhea virus (BVDV) free, unlike fetal lamb kidney (FLK) cells, currently used for this purpose. Following standardization, iELISA results were compared with those obtained by AGID and the commercial Chekit Leucose-Serum ELISA. Compared to AGID, iELISA had 94,44% sensitivity, 75.68% specificity, 79.10% positive predictive value (PPV) and 93.30% negative predictive value (NPV), with 84% concordance and a Kappa index of 0.699. Compared to the Chekit Leucose-Serum ELISA, iELISA showed 92.60% sensitivity, 87.09% specificity, 90.27% PPV and 90,00% NPV, with 90.27% concordance and a Kappa index of 0.801. Taking into account the high agreement with the traditional tests and the absence of non-specific reactions with BVDV, the developed assay could be used as diagnostic method to control EBL in Brazil.(AU)
A leucose enzoótica bovina (LEB) é uma doença infecciosa natural dos bovinos com distribuição mundial causada pelo "bovine leukemia virus" (BLV). A imunodifusão em gel de ágar (IDGA) foi considerada por muitos anos o teste de eleição, porém ensaios imunoenzimáticos (ELISA) apresentam sensibilidade mais elevada e leitura mais rápida e objetiva. No entanto, a importação de kits de ELISA é um processo dispendioso e demorado, e atualmente não há kits de IDGA comercialmente disponíveis no Brasil. Desta forma, o objetivo deste trabalho foi padronizar um ELISA indireto (iELISA) para diagnóstico da LEB utilizando antígenos produzidos a partir do cultivo do BLV em linhagem celular Tadarida brasiliensis "lung" (Tb1Lu) livre de "bovine viral diarrhea virus" (BVDV), diferentemente do que acontece com as linhagens "fetal lamb kidney" (FLK) atualmente utilizadas na produção desses antígenos para uso em ensaios sorológicos. Após a padronização do iELISA, os resultados foram comparados com aqueles obtidos por IDGA e pelo ELISA comercial "Chekit Leucose-Serum". Comparado ao IDGA, o iELISA apresentou 94,44% de sensibilidade, 75,68% de especificidade, valor preditivo positivo (VPP) de 79,1% e valor preditivo negativo (VPN) de 93,3%, com concordância entre os testes de 84% e o índice Kappa 0,699. Quando comparado ao ELISA "Chekit Leucose-Serum", o iELISA apresentou sensibilidade de 92,6%, especificidade de 87,09%, VPP de 90,27% e VPN de 90%, com concordância de 90,27% e o índice Kappa 0,801. Portanto, devido à alta concordância com os testes tradicionais e ausência da ocorrência de reações inespecíficas com BVDV, o ensaio desenvolvido pode ser utilizado como ferramenta diagnóstica para o controle da LEB no Brasil.(AU)
Subject(s)
Animals , Cattle , Cattle/virology , Enzyme-Linked Immunosorbent Assay/methods , Enzootic Bovine Leukosis/diagnosisABSTRACT
Background: Chronic Periodontitis (CP) is an infectious disease resulting in inflammation of supporting tissues of the teeth. A number of pro-inflammatory cytokines are formed against periodontopathogenic microorganisms. Interleukin-17 (IL-17) is a pro-inflammatory cytokine, implicated in numerous inflammatory and autoimmune conditions.Methods: A total of 25 periodontally healthy subjects (Group 1), 25 patients with gingivitis (Group 2) and 25 patients with CP (Group 3) were included for the study based on clinical examination. Gingival index, probing pocket depth and clinical attachment loss were recorded in all subjects.Results: The levels of IL-17 increased from healthy to gingivitis to periodontitis patients. A positive correlation was found with the IL-17 and the clinical parameters like gingival index, probing pocket depth and clinical attachment loss.Conclusions: There is a strong association between the levels of IL-17 with periodontal disease as well as with its severity and its possible use as a biomarker for inflammatory periodontal disease.