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@#At present,the most commonly used method for detecting hemagglutinin(HA)content in influenza vaccines is still single-radial immunodiffusion(SRID). However,the preparation of standards required by this method takes a long time,usually 2 ～ 3 months. Therefore,how to quantitatively analyze HA accurately has always been a difficult problem in the detection of HA content in the situation that reference products can not be obtained at the early stage of the pandemic influenza. High performance liquid chromatography(HPLC)has its own characteristics of rapidity,high sensitivity,good repeatability and high accuracy,which can rapidly determine HA content by using different separation principles and has been widely used in the detection of HA content in influenza vaccine. This paper reviewed the research progress of the application of HPLC in the determination of HA content in influenza vaccine.
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Introducción: La Inmunodifusión radial simple es una técnica con fundamento inmunológico confiable por su especificidad para la cuantificación de inmunoglobulinas principales y se emplea también para otras proteínas. Las placas de Inmunodifusión comerciales se ofertan con un número determinado de pocillos donde se coloca la muestra biológica que contiene la proteína a cuantificar. Objetivo: Evaluar la sensibilidad y la especificidad de la modificación introducida para optimizar el uso de las placas de inmunodifusión radial simple de la marca SIEMENS por aumento del número de muestras por placas. Material y Métodos: Se presenta una innovación que permite optimizar el área biológicamente activa de la placa no utilizada para emplearla para la cuantificación de otras muestras. Se realizan montajes paralelos de muestras de controles en los pocillos tradicionales y en los realizados en los espacios disponibles para cuantificar IgG y albúmina para suero y líquido cefalorraquídeo. Resultados: La sensibilidad del empleo por el método tradicional y por el nuevo no presenta diferencias significativas. En cuanto a la especificidad tampoco existen diferencias significativas menos en las placas para cuantificar albúmina en suero por lo que se recomienda diluir la muestra de suero antes de ser utilizada en el área disponible. En el caso de las placas NOR y LC Partigen® el número de muestras a ser beneficiadas con la cuantificación se duplica, pero de igual manera puede ser aplicada en otras placas de otras firmas comerciales. Conclusiones: Esta innovación permite hacer un uso óptimo de las placas de inmunodifusión con el consiguiente ahorro de material de importación y se puede aplicar fácilmente en todos los laboratorios del país(AU)
Introduction: Single radial immunodiffusion assay is a technique with immunological base, which is reliable because of its specificity in the quantification of main immunoglobulins, although it is also used for other proteins. Commercial immunodiffusion plates are offered with a determined number of holes where the biological samples containing protein to be quantified are placed. Objective: To evaluate the sensitivity and specificity of the modification implemented to optimize the usage of single radial immunodiffusion plates from Siemens by increasing the number of samples in the plates. Materials and Methods: An innovating procedure that allows to optimize the non-used biologically active area and use it in the quantification of other samples is presented. A parallel quantification of control samples from traditional holes and the other ones opened in available spaces was performed in order to quantify IgG and albumin in serum and in cerebrospinal fluid. Results: Sensitivity was not affected significantly between the normal plates and the usage of the new procedure. Regarding specificity, there are also no significant differences except in the plates used to quantify serum albumin; so, it is recommended to dilute serum samples before the application. In case of NOR and LC Partigens®, this proposed modification duplicates the number of samples to be quantified in each plate, but otherwise, it could be applied in other commercial immunoplates. Conclusions: This innovation allows to make an optimal usage of immunodiffusion plates with the consequent saving of import materials, which can be easily applied in all the laboratories of the country(AU)
Subject(s)
Humans , Laboratory Equipment , Immunodiffusion/methods , Mandatory TestingABSTRACT
Objective@#Quadrivalent influenza vaccines contain two lineages of type B virus, this study aimed to assess whether the result of single radial immunodiffusion (SRID) are accurate. The cross-interference of two type B hemagglutinins remains unknown.@*Methods@#We detected the vaccine samples developed by Jiangsu GDK Biological Technology Co., ltd by SRID.@*Results@#There was no significant difference between the HA content of antigen reagent, bulk sample and mixed sample of two B bulk within 10 to 40 μg/ml (P>0.05). Then each hemagglutinin B was diluted respectively by other three HA, 30 μg/ml, the other hemagglutinin B or phosphate buffer solution were measured within 10-160 μg/ml. Within 10-40 μg/ml, the HA content was proportional to the diameters of immunodiffusion (R2=0.998), while within the higher content range, a ternary linear regression equation fitted best (R2=0.999).@*Conclusions@#No cross-interference between B/Brisbane and B/Phuket was found in SRID within both detection ranges.
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Purpose: The management of burn patients is always challenging for the clinician due to high risk of bacterial sepsis, multi‑organ failure and death. Our objective was to study complement activation, C3 and interleukin‑6 (IL‑6) levels in burn patients and evaluate their role as prognostic markers. Materials and Methods: A total of 63 burn patients and 60 healthy controls were included in this study. Blood was collected from patients within 24 h and at 7th day of injury. Complement activation was determined by crossed electrophoresis and counter‑current immunoelectrophoresis. C3 levels were measured using a single radial immunodiffusion. IL‑6 was detected by ELISA. Results: All patients showed initial complement activation. Mean C3 levels showed an inverse correlation with the severity of burn. Patients with ≥20% burns had lower C3 than the controls (P < 0.001) and those with <20% burns (P < 0.001). Patients with ≥40% burns had activated complement and low C3 in 2nd week; they subsequently developed infection. Complement was inactive and C3 levels recovered in patients with <40% burns. The non‑survivors showed significantly lower C3 than the survivors (P < 0.05) in 2nd samples. Patients who developed infection had C3 significantly lower than those who remained free of infection (P < 0.05). All patients showed initial elevation in IL‑6 levels. Patients with ≥60% burns had significantly higher IL‑6 than controls (P < 0.001) and those with <60% burns (P < 0.001). Non‑survivors had higher IL‑6 than survivors in both samples (P < 0.001). Patients who developed infection showed significantly higher IL‑6 in 2nd samples than those without infection (P < 0.001). Conclusions: Complement activation, C3 and IL‑6 levels correlated well with the severity of injury and development of infection in burn patients. These parameters can be used to predict the onset of infection, septicaemia and mortality in burn patients.
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Objective To establish a method for rapid preparation of antiserum against influenza virus (H7N9) hemagglutinin,and to study the possibility of using it in single radial immunodiffusion (SRID) assay for quantitative detection of antigen in H7N9 influenza vaccine.Methods Hemagglutinin proteins expressed in eukaryotic cells were used to immunize sheep.Serum samples were collected to detect antibody titers by ELISA and double immunodiffusion assay.Different concentrations of antiserum were used in SRID assay to get the optimized concentration.Results After 4 times of immunization,the antiserum titers achieved 1 ∶ 1 000 000 and 1 ∶ 32 as indicated by ELISA and double immunodiffusion assay,respectively.The antiserum could form a clear precipitation line in SRID assay.The detection of antigen in the range of 10 to 40 μg/ml showed good linearity in the standard curve.The antigen titers in six batches of H7N9 vaccine detected by this SRID assay were identical with those by SDS-PAGE assay.Conclusion The antiserum against H7N9 hemagglutinin for SRID assay was developed successfully,and could be used as a reagent for the quantitative detection of antigen in H7N9 influenza vaccine.
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La transmisión de Brucella abortus a becerras de vacas positivas y negativas se determinó a la primera semana de vida y al tercer mes de edad. Se trabajó con dos hatos: el hato 1, con 670 vacas en producción, presentaba una seroprevalencia a brucelosis de 21.6% (145/670). En este hato se formaron dos grupos: vacas positivas y vacas negativas, como resultado de las pruebas de tarjeta y de inmunodifusión radial (IDR) realizadas con hapteno nativo. Se tomaron pruebas de sangre de las vacas en dos ocasiones, a la semana de edad y antes de que los animales fueran vacunados contra B. abortus. De las 22 vacas del grupo positivo, 2 (9.1%) becerras resultaron positivas a la primera semana de vida, pero no se encontraron vacas positivas a los tres meses de edad. En el grupo de becerras nacidas de vacas negativas no se encontraron animales positivos a la semana de vida, pero a los tres meses de edad, 4 de las 22 becerras resultaron positivas con la prueba de IDR. La tasa de prevalencia de vacas positivas a B. abortus fue de 13.6% a los tres meses de edad. De las 20 muestras de leche obtenidas de este hato se aisló B. abortus (100%). Mediante PCR se confirmó que estas cepas correspondían a cepas de campo y no a cepas vacunales. El hato 2, con 1800 vacas en producción, estaba inscrito en la campaña nacional contra la brucelosis animal y presentaba una seroprevalencia de 1.94% (35/1800) detectada de enero a diciembre de 2009. Se analizaron 1 170 registros usando los resultados de las pruebas de tarjeta y rivanol aplicada en becerras menores de tres meses de edad, de las que 24 (2.1%) resultaron positivas a B. abortus de enero de 2009 a junio de 2010. Se concluye que es necesario realizar el diagnóstico de brucelosis en becerras nacidas en establos donde se ha presentado la enfermedad, para prevenir que permanezcan animales positivos en el hato, ya que los anticuerpos posvacunales impedirán detectar la enfermedad, pero posteriormente se manifestará mediante abortos durante la primera gestación, perpetuando así la brucelosis en el establo.
Transmission of Brucella abortus to female calves from positive and negative cows was determined in the first week and third month of age. Two herds were used. Herd 1 consisted of 670 milking cows with a brucellosis seroprevalence of 21.6% (145/670). In this herd, groups of positive and negative cows were formed using the card and radial immunodifussion (RID) tests with native hapten. Blood samples were taken from female calves on two occasions: at one week of age and before animals were vaccinated against B. abortus. Of the 22 calves from the positive group, two (9.1%) were positive in the first week of life, but no more positive calves were found at three months of age. In the group of female calves born to negative cows, there were no positive animals at one week of age, but four out of 22 were found positive with the RID test at three months of age. A prevalence rate of 13.6% of positive calves for B. abortus in the third month of age was calculated. Twenty milk samples were obtained from this herd and B. abortus was isolated from all of them (100%). Using PCR, the strains found were confirmed to be field strains and not vaccine strains. Herd 2 consisted of 1800 milking cows, participating in the National Campaign against Animal Brucellosis, that had a seroprevalence of 1.94% (35/1800) detected from January to December 2009. In this herd, 1 170 records were analyzed using the results of the card and rivanol tests obtained from female calves younger than three months of age, of which 24 (2.1%) were found positive for B. abortus from January 2009 to June 2010. It is concluded that the diagnosis of brucellosis is necessary in female calves born in dairies to cows that have the disease, in order to prevent positive animals from remaining in the herd. Vaccine-induced antibodies will avert disease detection, but brucellosis will later manifest itself through abortions during first pregnancies, thus perpetuating the disease in dairies.
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Objective To study the possibility of using heterogeneous antiserum in single radial immunodiffusion (SRID) for quantitative detection of HA in H7N9 influenza vaccine product when H7N9-specific antiserum is not available in order to establish a testing method for the detection of H 7N9 antigen in any urgent situation.Methods Antisera specific for H7N1, H7N2, H7N3 and H7N7 were obtained from NIBSC and used for SRID assay .Amino acid sequences of hemagglutinins were comparatively analyzed be-tween H7N9 virus and other viruses used to prepare heterogeneous antiserum .The titers of antisera against H7N9 and their homogenous antigens were detected by double immunodiffusion method .Based on the results of homology analysis and cross-reaction, a suitable antiserum was selected and its applicability was further validated by the SRID assay using H7N9 antigen.Results Influenza A virus subtype H7N3 that used for preparation of 07/278 antiserum showed the highest HA homology with H7N9 (97.14%).The titer of 07/278 antiserum against H7N9 antigen was 1 ∶8 as detected by double immunodiffusion assay .The H7N9 anti-gen and the 07/278 antiserum could form a clear precipitation line in SRID assay .The detection of H7N9 antigen in the range of 10 to 40μg/ml showed a good linearity in the standard curve .Conclusion The 07/278 antiserum from NIBSC can be used as an alternative reagent for the quantitative detection of hemaggluti -nin in H7N9 influenza virus vaccine .
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Introducción: La apitoxina que es producida por la Apis mellifera posee efecto antiinflamatorio sobre una serie de marcadores biológicos. La prostaglandina E2 forma parte de ellos, estando presente en el fluido gingival crevicular (FGC). La prostaglandina E2 es evidenciada en la enfermedad periodontal. Objetivo: En este estudio se evaluó el efecto antiinflamatorio de la apitoxina sobre la concentración de prostaglandina E2 del FGC de un paciente sin enfermedad periodontal (SEP) y otro con enfermedad periodontal (CEP). Materiales y Método: Se seleccionó un paciente SEP y otro CEP, que sometidos a apiterapia durante 28 días, se registraron 5 muestras por paciente de FGC, siendo almacenadas, centrifugadas y refrigeradas para su conservación. Posteriormente se midió la concentración de prostaglandina E2 crevicular mediante inmunodifusión radial simple en placas petri con concentración de anticuerpo anti prostaglandina E2 de 1:1000. Selladas a 4°C, se esperó 72 horas para permitir su difusión, tiñéndose con Azul brillante de Coomasie, determinándose la concentración de cada placa. Resultados: Paciente SEP inmediatamente antes de apiterapia presentó una concentración de 0.9636 ± 0.0055 (ug/uL), finalizando con una concentración de 0.9196+/-0.0733 (ug/uL) al completar 28 días de tratamiento. El paciente CEP antes de recibir apiterapia presento una concentración de 1.1866 +/- 0.0867 (ug/uL), finalizando con una concentración de 0.9858 +/- 0.0074 (ug/uL) al completar 28 días de tratamiento. Discusión: Los hallazgos de este estudio demuestran una disminución de la concentración de PGE2 del FGC tanto para el paciente CEP y SEP sometidos a apiterapia durante 28 días, siendo esta disminución 3.7 veces mayor en el paciente CEP.
Introduction: Apitoxin, which is produced by Apis mellifera, has anti-inflammatory effect on a number of biomarkers. Prostaglandin E2 is one of them, being present in gingival crevicular fluid (GCF). Prostaglandin E2 is evidenced in periodontal disease. Objective: This study evaluated the antiinflammatory effect of apitoxin on concentration of prostaglandin E2 FGC in a patient with no periodontal disease (SEP) and other with periodontal disease (CEP). Materials and Methods: We selected both a SEP and CEP patient who were subjected to apitherapy for 28 days. There were 5 samples per patient of FGC, being stored, centrifuged and refrigerated for their preservation. Subsequently, the concentrations of crevicular prostaglandin E2 were measured by simple radial immunodiffusion in petri dishes with antibody concentration of prostaglandin E2 of 1:1000. Sealed at 4 °C, after 72 hours to allow diffusion, they were stained with Coomassie Brilliant Blue, determining the concentration of each plate. Results: SEP patient immediately before apitherapy presented a concentration of 0.9636 +/- 0.0055 (g / mL), ending with a concentration of 0.9196 +/- 0.0733 (g / mL) upon completion of 28 days of treatment. CEP patient before receiving apitherapy showed a concentration of 1.1866 +/- 0.0867 (g/mL), ending with a concentration of 0.9858 +/- 0.0074 (g/mL) upon completion of 28 days of treatment. Discussion: The findings of this study show a decrease in the concentration of PGE2 of FGC both for the CEP and SEP patient subjected to apitherapy for 28 days, being this decrease 3.7 times higher in the CEP patient.
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Humans , Adult , Anti-Inflammatory Agents , Bees , Dinoprostone/analysis , Periodontal Diseases/metabolism , Gingival Crevicular Fluid/chemistry , Apitherapy , Periodontal Diseases/therapy , Immunodiffusion , BiomarkersABSTRACT
PURPOSE: All IgG subclasses such as IgG1, IgG2, IgG3 and IgG4 can be transferred from mother to fetus through the placenta, though the amount of each IgG subclass is different from one another. Maternally acquired immunity might have an important role for the protection against the infections. We studied transplacental passage of IgG subclasses. METHODS: In this study, we observed the transplacental passage of IgG-subclasses in 22 paired samples of maternal and full- term fetal cord sera. Gestational ages varied from 37 to 42 weeks. The concentrations of IgG subclasses were analyzed by radial immunodiffusion method using commercialized Human IgG Subclass Combi kit. RESULTS: The concentrations of IgG subclasses, IgG1, IgG3 and IgG4 in cord sera exceed the maternal concentration, while IgG2 did not. The ratio of serum levels of cord to maternal were 1.330+/-0.067 for IgG1, 0.859+/-0.039 for IgG2, 1.258+/-0.058 for IgG3 and 1.159+/-0.038 for IgG4. CONCLUSION: This result suggested that the placenta may play a selective barrier for passage of IgG2.