Seminario biomol
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Transcript of Seminario biomol
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MARÍA CAMILA OSPINA JIMÉNEZSARA TORO CORREA
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INTRODUCTION
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VARICELLA ZOSTERGROUP: Group I (dsDNA)ORDER: HerpesviralesFAMILY: HerpesviridaeSUBFAMILY: AlphaherpesvirinaeGENUS: Varicellovirus.SPECIES: Human herpesvirus 3 (HHV-3)
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VARICELLA ZOSTER
Is a human alpha herpes virus witch causes varicella and herpes zoster (HZ) upon infeccion.
Primary infection with VZV causes varicella, while reaction latent virus results in HZ.
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VARICELLA ZOSTER
Varicella can be complicated by skin, pneumonia,
encephalomyelitis and myelitis.
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VARICELLA ZOSTERCatching chickenpox. It's also called the varicella
vaccine, because chickenpox is caused by
the varicella-zoster virus. The vaccine is made from a
live but weakened, or attenuated, virus.
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VARICELLA ZOSTER
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IMMUNITY
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ELISA
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VARICELA ZOSTER VIRUS
VIRUS ENTERS INTO THE CELL
PRESENCE OF CYTOKINES
ATTRACTION OF NEUTROPHILS AND
MACROPHAGESPHAGOCYTOSIS
ANTIGENS PRESENTATION TO A
T HELPER LYMPHOCYTES
T CYTOTOXIC LYMPHOCYTES AND B
LYMPHOCYTES RECOGNIZE THE
ANTIGEN
STIMULATION OF CELLULAR AND
HUMORAL IMMUNITY
T LYMPHCYTES HELPS B LYMPHOCYTES TO
PRODUCE ANTIBODYES
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OVERALL OBJECTIVE
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EVALUATE THE EFFECTIVENESS OF THE USE OF ELISA SANDWICH TESTS IN THE DETECTION OF IMMUNITY POSITIVE OR
NEGATIVE AGAINST THE VARICELLA ZOTER VIRUS
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MATERIALES Y METODOS
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MATERIALES Y METODOS: CELULAS, VIRUS, SUERO Y GLICOSIDASAS
Células de Spodoptera frugiperda Medio CCM3 con 2% de suero bovino fetal
Células epiteliales de pigmento retiniano humano Medio de Eagle modificado con 10% de FBS
Cepa vacuna oka ARPE 19
Vaculovirus Clontech
125 sueros humanos Beijing Wantai Biological Pharmacy Enterprise
240 sueros humanos Instituto Nacional de Diagnóstico y Desarrollo de Vacunas en Enfermedades infecciosas
Suero VZV positive calibrado Glicosidasa y Rnasa B = New Engkand Biolabs
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MATERIALES Y METODOS: EXPRESION Y PURIFICACION DE LA IgE
Fragmento gE marcado con His se expreso usando el vacuovirus
rgE agregado al sobrenadante del cultivo
Dos bandas se purificaron con una columna de Ni – NTA
Separacion de bandas por columna de flujo rápido de DEAE
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MATERIALES Y METODOS: ANALISIS DE GLICOSILACION
Proteina igE purificada y
Rnasa B
• Digestion con péptido N glicosidasa F (PNGasa F), endoglicosidasa H (Endo H) u O glicosidasa y neuroaminidasa.
Analisis • SDS- PAGE y coloración azul brillante de Coomasie.
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MATERIALES Y METODOS: ELISA INDIRECTO BASADO EN GPS VZV (GP
ELISA) Y gE (gE ELISA)Fijación de
antígenos al soporte
insoluble. Lavado
Adición del suero
problema con anticuerpos.
Lavado
Adición de anti-
anticuerpos conjugados
con una enzima
Adición de substrato sobre el cual pueda actuar la enzima marcadora
Lectura visual o colorimétrica
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MATERIALES Y METODOS: ELISA INDIRECTO BASADO EN GPS VZV (GP
ELISA) Y gE (gE ELISA)Se emplea en ensayos de competición de antígeno
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ELISA DOBLE DE ANTIGENO gE
Fijación de soporte
insoluble de anticuerpos
específicos del agente
patógeno a detectar
Adición de la muestra
problema que contiene el
agente patógeno
diagnostico.
Adicion de anticuerpos
específicos del antígeno a detectar (epitopo
diferente).
Adicion de anti cuerpos
conjugados con una enzima
anti-anticuerpos .
Adicion del substrato y lectura de resultados.
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ELISA DOBLE DE ANTIGENO gE
Detección de infecciones. Ayuda diagnostica.
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PRUEBA FAMA
Validada inicialmente como un indicador especifico de VZV.
Células infectadas con V-OKA fueron recolectadas cuando el efecto citopatico estaba presente en aproximadamente 70-80% de las células.
Después de varios procesos se colocaron en un portaobjetos y se incubaron con dos sueros humanos diluidos.
Isotiocianato de fluoresceína (FITC) conjugado de cabra anti IgG humano pre-mezclado se incubo con las células junto con Azul de Evans y se observo al microscopio.
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RESULTADOS
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EXPRESSION, PURIFICATION AND ANALYSIS OF RECOMBINANT gE PROTEIN
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PERFORMANCE OF gpELISA, gE ELISA, AND DOUBLE gE ANTIGEN SANDWICH ELISA IN THE DETECTION OF HUMAN VZV POSITIVE AND VZV NEGATIVE SERUM
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CHARACTERISTICS OF THE DOUBLE gE ANTIGEN SANDWICH ELISA IN HUMAN SERUM DETECTION
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EVALUATION OF HUMAN SERUM USING DE DOUBLE gE ANTIGEN SANDWICH ELISA COMPARED TO THE FAMA TEST AND A COMMERCIAL ELISA KIT
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QUANTITATIVE EVALUATION OF
POPULATION INMUNITY TO VZV
USING THE ESTABLISHED DOUBLE gE ANTIGEN
SÁNDWICH ELISA
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DISCUSSION
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Author Yes or not
Guris et al. 2008
“Immunization with varicellavaccine has dramatically reduced
the incidence of varicella”.
Keller et al. 1986
“VZV-positive sera have been shown to contain a high
titre of gE-specific antibodies”
Sauerbrei et al.2012
" Commercial tests adopt a similar indirect ELISA format but use
different coating antigens"
Wasmuth and Miller 1990
"The effective antigen in VZV gps varies from batches and should be
standardized with an in-house panel serum"
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CONCLUSION
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The vaccine for chickenpox has decreased the incidence
of varicella in many countries. However, in many others it is
not included within the vaccination scheme and it is
necessary to change this aspect to decrease the
number of patients affected and the complications that
the virus generates.
The varicella virus has the ability to trigger processes of
cellular and humoral immunity in the body and its
main marker is the IgE antibody used for testing.
FAMA test continues being the main for to evaluate the immunity that the body has against the varicella zoster
virus
The double gE antigen sandwich ELISA can be used
to evaluate who has immunity to vzv, because this test was demonstrated to be highly specific to detect negative
sera.
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MAPA CONCEPTUAL DE SARA TORO
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MAPA CONCEPTUAL DE MARÍA CAMILA OSPINA
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GRACIAS POR SU ATENCIÓN