Name: a protein microarray assay for serological determination of antigen-specific antibody responses following clostridium difficile infection
Uploaded: 2018-06-15T14:00:00
Description: Watch this Scientific Journal Video about A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection at JoVE.com

Esta investigación fue apoyada por una beca de Hermes a Ola Negm y Tanya Monaghan y a través de fondos separados del centro de enfermedades digestivas de Nottingham y el NIHR Nottingham digestivas enfermedades centro de investigación biomédica.

1. preparar una placa de microchips
<ol><li>Diluir los antígenos de C. difficile con un tampón de impresión [PBS-Tween-trehalosa (50 mM)] a la concentración óptima (que estaba predefinida antes de ejecutar el suero del paciente): toxina (200 μg/mL) y la toxina B (100 μg/mL), pCDTb (200 μg/mL) y natural purificada SLP todo deriva de Benito 001, 002 y 027 (200 μg/mL). Nota: La toxina B se obtuvo de una toxina B-expresión cepa CCUG 20309 (90 μg/mL).<ol>
<li>Diluir los controles positivos, lisados de...

<ol>
	<li>Negm, O. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+humoral+immune+responses+to+Clostridium+difficile.-specific+antigens+by+protein+microarray+analysis.">Profiling humoral immune responses to Clostridium difficile.-specific antigens by protein microarray analysis.</a> Clinical and Vaccine Immunology. 22 (9), 1033-1039 (2015).</li><li>Monaghan, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+prevalence+of+subclass-specific+binding+and+neutralizing+antibodies+against+Clostridium+difficile.+toxins+in+adult+cystic+fibrosis+sera:+possible+mode+of+immunoprotection+against+symptomatic+C.+difficile+infection.">High prevalence of subclass-specific binding and neutralizing antibodies against Clostridium difficile. toxins in adult cystic fibrosis sera: possible mode of immunoprotection against symptomatic C. difficile infection.</a> Clinical and Experimental Gastroenterology. 10, 169-175 (2017).</li><li>Negm, O. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+antibodies+against+Clostridium+difficile.+are+present+in+intravenous+immunoglobulin+and+are+retained+in+humans+following+its+administration.">Protective antibodies against Clostridium difficile. are present in intravenous immunoglobulin and are retained in humans following its administration.</a> Clinical &amp; Experimental Immunology. 188 (3), 437-443 (2017).</li><li>Monaghan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OC-058+A+protein+microarray+assay+for+predicting+severe+outcomes+in+Clostridium+difficile+infection.">OC-058 A protein microarray assay for predicting severe outcomes in Clostridium difficile infection.</a> Gut. 66, 31 (2017).</li><li>Bacon, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+G+directed+against+toxins+A+and+B+of+Clostridium+difficile+in+the+general+population+and+patients+with+antibiotic-associated+diarrhea.">Immunoglobulin G directed against toxins A and B of Clostridium difficile in the general population and patients with antibiotic-associated diarrhea.</a> Diagnostic Microbiology and Infectious Disease. 18 (4), 205-209 (1994).</li><li>Viscidi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+antibody+response+to+toxins+A+and+B+of+Clostridium+difficile.">Serum antibody response to toxins A and B of Clostridium difficile.</a> The Journal of Infectious Diseases. 148 (1), 93-100 (1983).</li><li>Salcedo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+immunoglobulin+therapy+for+severe+Clostridium+difficile+colitis.">Intravenous immunoglobulin therapy for severe Clostridium difficile colitis.</a> Gut. 41 (3), 366-370 (1997).</li><li>Abougergi, M. S., Kwon, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+immunoglobulin+for+the+treatment+of+Clostridium+difficile+infection:+a+review.">Intravenous immunoglobulin for the treatment of Clostridium difficile infection: a review.</a> Digestive Diseases and Sciences. 56 (1), 19-26 (2011).</li><li>Shah, N., Shaaban, H., Spira, R., Slim, J., Boghossian, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+immunoglobulin+in+the+treatment+of+severe+Clostridium+difficile+colitis.">Intravenous immunoglobulin in the treatment of severe Clostridium difficile colitis.</a> Journal of Global Infectious Diseases. 6 (2), 82-85 (2014).</li><li>Kyne, L., Warny, M., Qamar, A., Kelly, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymptomatic+carriage+of+Clostridium+difficile+and+serum+levels+of+IgG+antibody+against+toxin+A.">Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A.</a> The New England Journal of Medicine. 342 (6), 390-397 (2000).</li><li>Solomon, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+in+patients+with+Clostridium+difficile+infection+correlates+with+host+pro-inflammatory+and+humoral+immune+responses.">Mortality in patients with Clostridium difficile infection correlates with host pro-inflammatory and humoral immune responses.</a> Journal of Medical Microbiology. 62, 1453-1460 (2013).</li><li>Kyne, L., Warny, M., Qamar, A., Kelly, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+antibody+response+to+toxin+A+and+protection+against+recurrent+Clostridium+difficile+diarrhoea.">Association between antibody response to toxin A and protection against recurrent Clostridium difficile diarrhoea.</a> Lancet. 357 (9251), 189-193 (2001).</li><li>Leav, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+anti-toxin+B+antibody+correlates+with+protection+from+recurrent+Clostridium+difficile+infection+(CDI).">Serum anti-toxin B antibody correlates with protection from recurrent Clostridium difficile infection (CDI).</a> Vaccine. 28 (4), 965-969 (2010).</li><li>Drudy, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+antibody+response+to+surface+layer+proteins+in+Clostridium+difficile+infection.">Human antibody response to surface layer proteins in Clostridium difficile infection.</a> FEMS Immunology and Medical Microbiology. 41 (3), 237-242 (2004).</li><li>Bauer, M. P., Nibbering, P. H., Poxton, I. R., Kuijper, E. J., van Dissel, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humoral+immune+response+as+predictor+of+recurrence+in+Clostridium+difficile+infection.">Humoral immune response as predictor of recurrence in Clostridium difficile infection.</a> Clinical Microbiology and Infection. 20 (12), 1323-1328 (2014).</li><li>Wilcox, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bezlotoxumab+for+prevention+of+recurrent+Clostridium+difficile+infection.">Bezlotoxumab for prevention of recurrent Clostridium difficile infection.</a> The New England Journal of Medicine. 376 (4), 305-317 (2017).</li><li>Leuzzi, R., Adamo, R., Scarselli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccines+against+Clostridium+difficile.">Vaccines against Clostridium difficile.</a> Human Vaccines &amp; Immunotherapeutics. 10 (6), 1466-1477 (2014).</li><li>Ghose, C., Kelly, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prospect+for+vaccines+to+prevent+Clostridium+difficile+infection.">The prospect for vaccines to prevent Clostridium difficile infection.</a> Infectious Disease Clinics of North America. 29 (1), 145-162 (2015).</li><li>Henderson, M., Bragg, A., Fahim, G., Shah, M., Hermes-DeSantis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+safety+and+efficacy+of+vaccines+as+prophylaxis+for+Clostridium+difficile+infections.">A review of the safety and efficacy of vaccines as prophylaxis for Clostridium difficile infections.</a> Vaccines. 5 (3), (2017).</li><li>Zhu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+protein+activities+using+proteome+chips.">Global analysis of protein activities using proteome chips.</a> Science. 293 (5537), 2101-2105 (2001).</li><li>Delehanty, J. B., Ligler, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+printing+functional+protein+microarrays.">Method for printing functional protein microarrays.</a> BioTechniques. 34 (2), 380-385 (2003).</li><li>Sutandy, F. X., Qian, J., Chen, C. S., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+protein+microarrays.">Overview of protein microarrays.</a> Current Protocols in Protein Science. , 21 (2013).</li><li>Huang, Y., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+array-based+approaches+for+biomarker+discovery+in+cancer.">Protein array-based approaches for biomarker discovery in cancer.</a> Genomics, Proteomics &amp; Bioinformatics. 15 (2), 73-81 (2017).</li><li>Vigil, A., Davies, D. H., Felgner, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+humoral+immune+response+to+infectious+agents+using+high-density+protein+microarrays.">Defining the humoral immune response to infectious agents using high-density protein microarrays.</a> Future Microbiology. 5 (2), 241-251 (2010).</li><li>Bacarese-Hamilton, T., Mezzasoma, L., Ardizzoni, A., Bistoni, F., Crisanti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serodiagnosis+of+infectious+diseases+with+antigen+microarrays.">Serodiagnosis of infectious diseases with antigen microarrays.</a> Journal of Applied Microbiology. 96 (1), 10-17 (2004).</li><li>Davies, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+the+humoral+immune+response+to+infection+by+using+proteome+microarrays:+high-throughput+vaccine+and+diagnostic+antigen+discovery.">Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (3), 547-552 (2005).</li><li>Wadia, P. P., Sahaf, B., Miklos, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+antigen+microarrays+for+serum/plasma+antibody+detection.">Recombinant antigen microarrays for serum/plasma antibody detection.</a> Methods in Molecular Biology. 723, 81-104 (2011).</li><li>Moore, C. D., Ajala, O. Z., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+in+high-content+functional+protein+microarrays.">Applications in high-content functional protein microarrays.</a> Current Opinion in Chemical Biology. 30, 21-27 (2016).</li><li>Crompton, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective+analysis+of+the+Ab+response+to+Plasmodium+falciparum+before+and+after+a+malaria+season+by+protein+microarray.">A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (15), 6958-6963 (2010).</li><li>Khodakov, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+oligonucleotide+microarray+for+multiplex+real-time+PCR+identification+of+HIV-1,+HBV,+and+HCV.">An oligonucleotide microarray for multiplex real-time PCR identification of HIV-1, HBV, and HCV.</a> BioTechniques. 44 (2), 241-246 (2008).</li><li>Ryabinin, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+oligonucleotide+microarray+for+sub-typing+of+Influenza+A+virus.">Universal oligonucleotide microarray for sub-typing of Influenza A virus.</a> PLoS One. 6 (4), 17529 (2011).</li><li>Zhu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+acute+respiratory+syndrome+diagnostics+using+a+coronavirus+protein+microarray.">Severe acute respiratory syndrome diagnostics using a coronavirus protein microarray.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (11), 4011-4016 (2006).</li><li>Cleton, N. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spot+the+difference:+development+of+a+syndrome+based+protein+microarray+for+specific+serological+detection+of+multiple+flavivirus+infections+in+travelers.">Spot the difference: development of a syndrome based protein microarray for specific serological detection of multiple flavivirus infections in travelers.</a> PloS Neglected Tropical Diseases. 9 (3), 0003580 (2015).</li><li>Liu, Y., Yu, F., Huang, H., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+recombinant+antigen+array+for+simultaneous+detection+of+viral+antibodies.">Development of recombinant antigen array for simultaneous detection of viral antibodies.</a> PLoS One. 8 (9), 73842 (2013).</li><li>Zhou, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+array+identification+of+protein+markers+for+serodiagnosis+of+Mycobacterium+tuberculosis+infection.">Protein array identification of protein markers for serodiagnosis of Mycobacterium tuberculosis infection.</a> Scientific Reports. 5, 15349 (2015).</li><li>Mannsperger, H. A., Gade, S., Henjes, F., Beissbarth, T., Korf, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPPanalyzer:+analysis+of+reverse-phase+protein+array+data.">RPPanalyzer: analysis of reverse-phase protein array data.</a> Bioinformatics. 26 (17), 2202-2203 (2010).</li><li>Monaghan, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+antibody+and+memory+B-cell+responses+to+C.+difficile-associated+diarrhoea,+inflammatory+bowel+disease+and+cystic+fibrosis.">Circulating antibody and memory B-cell responses to C. difficile-associated diarrhoea, inflammatory bowel disease and cystic fibrosis.</a> PLoS One. 8 (9), 74452 (2013).</li><li>Grainger, D. W., Greef, C. H., Gong, P., Lochhead, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+microarray+surface+chemistries.">Current microarray surface chemistries.</a> Methods in Molecular Biology. 381, 37-75 (2007).</li><li>Wellhausen, R., Seitz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facing+current+quantification+challenges+in+protein+microarrays.">Facing current quantification challenges in protein microarrays.</a> Journal of Biomedicine and Biotechnology. 2012, (2012).</li><li>Guo, A., Zhu, X. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+critical+role+of+surface+chemistry+in+protein+microarrays.">The critical role of surface chemistry in protein microarrays.</a> Functional Protein Microarrays in Drug Discovery. , (2007).</li></ol>

En este protocolo, hemos demostrado que eso microarray es una plataforma adecuada para la definición de respuesta inmune humoral a antígenos de la proteína de C. difficile en el suero del paciente (figuras 3 y 6) y preparados comerciales de IgIV (figura 5). También hemos demostrado que la técnica de microarray realiza bien en comparación con ELISA convencional (figura 2) y muestra excelente reproducibilidad, intra...

Este protocolo describe el desarrollo y validación de un ensayo de microarray de proteínas novedosas y personalizadas para la detección y semi-cuantificación de cepa bacteriana y las respuestas de anticuerpos isotipo-específicos a los antígenos de C. difficile . Utilizamos con éxito nuestro C. difficile-análisis de microarrays específicas como una nueva herramienta prometedora para el Bioanálisis composición de contenido de anticuerpos específicos en el suero del paciente1,2, preparaciones de IVIg3, y identificación de especificidades de anticuerpos que se correlacionan con resultados deficientes en el CDI4. Demostramos cómo biobanked sueros y preparados comerciales de IVIg pueden ser analizadas en microarray diapositivas, permitiendo alta calidad reproducible de perfiles de las respuestas de anticuerpos patógenos específicos de C. difficile en este ensayo.
Muchos niños y adultos sanos tienen anticuerpos IgG e IgA sérica detectable las toxinas de C. difficile A y B5,6. Éstos se piensan para presentarse después de la exposición transitoria durante la infancia y después de la exposición a C. difficile en la edad adulta. Por esta razón, inmunoglobulina policlonal intravenosa ha sido utilizado apagado-etiquete para tratar recurrentes y fulminante CDI7,8,9. Sin embargo, su papel definitivo y modo de acción está claro. Varios estudios han demostrado que la respuesta inmunitaria humoral a las toxinas de C. difficile desempeña un papel en la presentación de la enfermedad y el resultado. En concreto, pacientes asintomáticos muestran una mayor concentración del suero antitoxina A IgG en comparación con los pacientes que desarrollan enfermedad sintomática10. Una asociación demostrable se ha divulgado para mediana antitoxina IgG A títulos y todas las causas mortalidad a 30 días11. Varios informes también han revelado una asociación con una protección contra respuestas recurrencia y anticuerpos a la toxina A, B y varios antígenos no toxina (Cwp66, Cwp84, FliC, FliD y proteínas de la capa superficial (SLPs))12,13 , 14 , 15. estas observaciones han estimulado el desarrollo de la primera inmunoterapia pasiva de drogas dirigida a C. difficile toxina B (bezlotuxumab), que recientemente ha sido aprobado por el alimento y administración de drogas y los medicamentos europeos Agencia para la prevención de la recurrente de la CDI16. Estrategias de vacunación con toxinas inactivadas o fragmentos de toxina recombinante también están actualmente bajo desarrollo17,18,19. Estos nuevos enfoques terapéuticos sin duda estimulará el requisito para la evaluación de la respuesta inmune humoral a múltiples antígenos en tamaños de muestra grandes.
Hoy en día, hay una notable falta de ensayos de alto rendimiento disponibles en el mercado capaz de evaluar simultáneamente la cepa bacteriana y las respuestas de anticuerpos isotipo-específicos a los antígenos de C. difficile . Hay una necesidad para el desarrollo de estos ensayos para facilitar esfuerzos de futuras investigaciones y aplicaciones clínicas. Microarrays de la proteína son un método para inmovilizar grandes cantidades de proteínas purificadas individualmente como una matriz espacial organizada de puntos sobre una superficie microscópica basada en diapositivas utilizando un sistema robótico, que puede ser un20 de contacto o sin contacto herramienta21de la impresión. Los puntos pueden representar mezclas complejas como los lysates de la célula, los anticuerpos, homogenados de tejido, proteínas endógenas o recombinantes o péptidos, fluidos corporales o drogas22,23.
Tecnología de microarrays de proteínas ofrece distintas ventajas sobre ELISA interna estándar técnicas, que tradicionalmente se han utilizado para evaluar las respuestas de anticuerpos de anti -C. difficile . Estos incluyen un aumento de la capacidad para detectar una gama de los multi-isotipo-específicos anticuerpos contra un panel más amplio de objetivos de la proteína, volumen reducido requisitos para antígenos, las muestras y reactivos y una mayor capacidad para incorporar una mayor número de repeticiones técnicas, además de múltiples control interno de calidad (QC) mide1. Microarrays son por lo tanto más sensibles, precisos y reproducibles y tener un mayor rango dinámico. Estos factores hacen microarrays una alternativa más barata y potencialmente favorable a ELISA para la detección a gran escala de las proteínas conocidas. Sin embargo, desventajas de la tecnología de microarrays resultan principalmente de los grandes costos iniciales asociados con establecer un panel de antígenos altamente purificados y configuración de la plataforma tecnológica.
Microarrays de la proteína se han utilizado en las últimas dos décadas como una herramienta de diagnóstico investigación básica en aplicaciones clínicas. Aplicaciones específicas incluyen la expresión de la proteína de perfiles, el estudio de las relaciones enzima-sustrato, investigación de biomarcadores, análisis de las interacciones huésped-microbio y perfiles de anticuerpos de especificidad de23,24, 2526,de,27,28. Se han establecido muchos nuevos microarrays de proteínas antígeno patógeno, incluyendo la malaria (Plasmodium)29,30de VIH-1, influenza31, síndrome respiratorio agudo severo (SARS)32, fiebre hemorrágica viral33 , herpesviruses34y35de la tuberculosis.
El presente Protocolo se relaciona con el establecimiento de un fácil funcionamiento C. difficile reactivo antígeno microarray ensayo, que permite la cuantificación exacta, precisa y específica de multi-isotipo y las respuestas de anticuerpos específicos de cepa a C. difficile antígenos en suero e inmunoglobulina intravenosa policlonal. En este documento, se incluyen resultados representativos pertenecientes a un funcionamiento de ensayo de microarray aceptable comparado con ELISA, así como análisis de precisión y reproducibilidad perfiles. Este análisis podrían desarrollarse aún más para otras muestras clínicas de perfil y establece un nuevo estándar para la investigación en bases moleculares de la CDI.

<table border="0" cellpadding="0" cellspacing="0" style="width:1827px;" width="1826">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">BioRobotics MicroGrid II arrayer</td>
			<td style="width:296px;">Digilab, Malborough, MA, USA</td>
			<td style="width:296px;">N/A</td>
			<td style="width:836px;">Contact arrayer used to automated spotting of the antigens onto the slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scanner InnoScan 710.</td>
			<td>Innopsys</td>
			<td>N/A</td>
			<td>A fluorescent microarray slide scanner with a red (Cy5) laser to read the reaction.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAPIX software version 7.2.0</td>
			<td>Innopsys</td>
			<td>N/A</td>
			<td>Measure signal intensities of the spots.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicon contact pin</td>
			<td>Parallel Synthesis Technologies</td>
			<td>SMT-P75</td>
			<td>Print the samples onto the slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific Nalgene Desiccator</td>
			<td>Thermo Scientific</td>
			<td>41102426</td>
			<td>To store the new and printed slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384-well plate</td>
			<td>Genetix</td>
			<td>X7022UN</td>
			<td>To prepare the antigens.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate cover</td>
			<td>Sigma Aldrich, UK</td>
			<td>CLS6570-100EA</td>
			<td>To reduce evaporation of the samples.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aminosilane slides</td>
			<td>Schott,&#160; Germany</td>
			<td>1064875</td>
			<td>The slide of choice for printing the antigens.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide holders</td>
			<td>GraceBio Labs, USA</td>
			<td>204862</td>
			<td>Divide the slides into identical 16 subarrays. These holders are re-usable, removable, leak-proof wells .&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Candida albicans lysate</td>
			<td>NIBSC</td>
			<td>PR-BA117-S</td>
			<td>Positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetanus Toxoid&#160;</td>
			<td>Athens Research and Technology</td>
			<td>04/150</td>
			<td>Positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G (IgG), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707</td>
			<td>Purified Native Human Immunoglobulin G IgG, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G1 (IgG1), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-1</td>
			<td>Purified Native Human Immunoglobulin G1 IgG1, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G2 (IgG2), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-2</td>
			<td>Purified Native Human Immunoglobulin G2 IgG2, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G3 (IgG3), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-3</td>
			<td>Purified Native Human Immunoglobulin G3 IgG3, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G4 (IgG4), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-4</td>
			<td>Purified Native Human Immunoglobulin G4 IgG4, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin A (IgA), Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090701</td>
			<td>Purified Native Human Immunoglobulin A (IgA), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin A1 (IgA1), Human Myeloma Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090701-1M</td>
			<td>Purified Native Human Immunoglobulin A1 (IgA1), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin A2 (IgA2), Human Myeloma Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090701-2M</td>
			<td>Purified Native Human Immunoglobulin A2 (IgA2), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin M (IgM), Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090713</td>
			<td>Purified Native Human Immunoglobulin M (IgM), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated Goat Anti-Human IgG Antibody, gamma chain specific</td>
			<td>Vector Labs</td>
			<td>BA-3080</td>
			<td>Goat anti- human IgG (&#947;-chain specific)-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG1 Hinge-BIOT</td>
			<td>Southern Biotec</td>
			<td>9052-08&#160;</td>
			<td>Goat anti- human IgG1 biotin antibody reacts specifically with human IgG1 but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG2 Fc-BIOT</td>
			<td>Southern Biotec</td>
			<td>9060-08&#160;&#160;</td>
			<td>Goat anti- human IgG2 -biotin antibody reacts specifically with human IgG 2but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG3 Hinge-BIOT</td>
			<td>Southern Biotec</td>
			<td>9210-08&#160;&#160;&#160;</td>
			<td>Goat anti- human IgG3-biotin antibody reacts specifically with human IgG3 but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG4 pFc&#39;-BIOT</td>
			<td>Southern Biotec</td>
			<td>9190-08&#160;&#160;</td>
			<td>Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Human IgA, alpha chain specific, made in goat - Biotinylated</td>
			<td>Vector Labs</td>
			<td>BA-3030</td>
			<td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgA1-BIOT</td>
			<td>Southern Biotec</td>
			<td>9130-08</td>
			<td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgA2-BIOT</td>
			<td>Southern Biotec</td>
			<td>9140-08&#160;&#160;</td>
			<td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgM-BIOT</td>
			<td>Southern Biotec</td>
			<td>9020-08&#160;</td>
			<td>Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 mm syringe filter</td>
			<td>Thermo scientific</td>
			<td>723-2520</td>
			<td>Filter the 5% BSA.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich, UK</td>
			<td>A7284</td>
			<td>Use 5% BSA for blocking the slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibody diluent</td>
			<td>Dako, UK</td>
			<td>S3022</td>
			<td>To dilute the serum and the secondary antibody.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Cy5</td>
			<td>eBioscience</td>
			<td>SA1011</td>
			<td>Detection of the immune reaction.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087)</td>
			<td>Toxins Group, Public Health England</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified whole C. difficile toxin B (CCUG 20309 toxin B only expressing strain)</td>
			<td>Toxins Group, Public Health England</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precursor form of B fragment of binary toxin, pCDTb</td>
			<td>University of Bath&#160;</td>
			<td>NA</td>
			<td>Produced in E. Coli from wholly synthetic recombinant gene construct. Amino acid sequence based on published sequence from 027 ribotype (http:www.uniprot.org/uniprot/A8DS70)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified native whole ribotype-specific (001, 002, 027) surface layer proteins</td>
			<td>Dublin City University&#160;</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vigam (IVIg preparation 1)</td>
			<td>Nottingham University Hospitals NHS Trust</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Privigen (IVIg preparation 2)</td>
			<td>Nottingham University Hospitals NHS Trust</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Intratect (IVIg preparation 3)</td>
			<td>Nottingham University Hospitals NHS Trust</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protein microarray assay for serological determination of antigen-specific antibody responses following clostridium difficile infection

Le ofrecemos una descripción detallada de un análisis de microarray de la novela de alto rendimiento de proteína para la determinación de anti -Clostridium difficile los niveles de anticuerpos en suero humano y en preparaciones separadas de inmunoglobulina policlonal intravenosa (IgIV). El protocolo describe los pasos metodológicos involucrados en la preparación de muestras, impresión de matrices, procedimiento de ensayo y análisis de datos. Además, este protocolo podría ser desarrollado para incorporar diversas muestras clínicas incluyendo plasma y sobrenadantes de cultivo de células. Mostramos cómo microarrays de la proteína se pueden utilizar para determinar una combinación de isotipo (IgG, IgA, IgM), subclase (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), y anticuerpos específicos de cepa altamente purificaron toda C. difficile toxinas A y B (toxinotype 0, tensión VPI 10463, ribotipo 087), la toxina B de una C. difficile toxina B-sólo expresando su cepa (CCUG 20309), una forma precursora de un fragmento de B de la toxina binaria, pCDTb, proteínas específicas ribotype de toda la superficie de la capa (PHL, 001, 002, 027) y control de las proteínas (tétanos tetánico y Candida albicans). Durante el experimento, microarrays están sondeados con sueros de individuos con infección por C. difficile (CDI), las personas con fibrosis quística (FQ) sin diarrea, controles sanos (HC) y de individuos pre- y post-IgIV tratamiento para la tratamiento de la CDI, el trastorno de inmunodeficiencia combinada y Polirradiculopatía desmielinizante inflamatoria crónica. Encontramos diferencias significativas en eficacia de neutralización de la toxina y los niveles de anticuerpos específicos de isotipo multi entre los grupos de pacientes, preparados comerciales de IVIg y sera antes y después administración de IVIg. También, hay una correlación significativa entre el microarray y análisis enzima-ligado del inmunosorbente (ELISA) para los niveles de antitoxina IgG en muestras de suero. Estos resultados sugieren eso microarray podría convertirse en una herramienta prometedora para perfilar las respuestas del anticuerpo a los antígenos de C.difficile en seres humanos infectados o vacunados. Con el mayor refinamiento de los paneles de antígeno y una reducción en los costos de producción, esperamos que la tecnología de microarrays puede ayudar a optimizar y seleccionar las inmunoterapias más clínicamente útiles para la infección de C. difficile en un modo específico para cada paciente.

La figura 1 muestra un diagrama de flujo que describe los principales pasos en el protocolo descrito. La figura 2 muestra pruebas de correlación de Spearman demostrar acuerdo entre microarray y ELISA para IgG e IgA los niveles A y B en los sueros de pacientes de prueba. La figura 3 muestra diferencial IgG e IgA anticuerpos clase las respuestas de anticuerpos específicos a la toxina A y toxina B tox...

Watch this Scientific Journal Video about A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection at JoVE.com

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

Este artículo describe un método de microarrays de la proteína simple para perfiles de respuesta inmune humoral a un panel de 7-plex de altamente purificaron antígenos de Clostridium difficile en suero humano. El protocolo puede ampliarse para la determinación de las respuestas de anticuerpos específicos en los preparados de inmunoglobulina intravenosa policlonal.

Un análisis de Microarray de proteínas para la determinación serológica de anticuerpos las respuestas siguientes infección por Clostridium difficile específica de antígeno

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

This method may help with optimizing and selecting clinically useful immunotherapies for Clostridium difficile infection in a patient-specific manner, and elucidating human immune responses to Clostridium difficile. The main advantage of this technique is that it enables accurate and simultaneous quantification of multi isotype and strain-specific antibody responses to C.difficile antigens. Generally, individuals that are new to this method will struggle because expertise is required in establishing and optimizing a panel of highly purified antigens, and setting up the technological platform.Visual demonstration of this method is critical, as methodological list types involved in preparing the microwave plate, arrays printing, a safe procedure, and the data analysis require attention to the details. Demonstrating some of the procedure will be by Sam Greenwood, a technician from my laboratory. To begin, dilute Clostridium difficile antigens and printing buffer to optimum concentration.Next, transfer 10 microliters of antigen solution to a 384 well plate. Cover the plate with a plate seal, and centrifuge at 300 times gravity for five minutes at room temperature. For microarray printing, use a hand-held gas burner to heat the silicon PIN three times for two seconds each.Then, rinse the silicon PIN in clean water three times for three seconds each. Place the microarray plate in the loading cartridge of an arrayer. Eject the tray, and then place the amino silane slides in the slide tray and press the OK button to turn the vacuum on.Insure that all of the slides are secured, and then click OK.Return the tray to the origin, and close the lid. Next, launch the appropriate program and go to File, then Microarraying Parameters, then Open. Select Clostridium difficile file for printing all antigens in quadruplicate.Under this window, open the Source tab to indicate sample storage, and Target tab to input microarray details. Then, open the Options tab to select the washing tool. Under the General tab of Run Preferences, on the TAS applications suite toolbar, select Wash at start of run, and Wash at end of run.Open the Climate tab, and set the humidity level between 55 to 60%Now start the run, and periodically check the arrayer to insure proper functioning. After printing, carefully place the printed slides in a slide holder with 16 multi-well chambers. Then add 100 microliters of 5%BSAT to each block.Cover the slides and incubate for one hour at room temperature with shaking. Use 120 microliters of PBST to wash each block five times for one minute each with shaking. Next, thaw out the serum samples obtained from C difficile infected patients and healthy controls on ice for 20 minutes.Add commercially available antibody diluent to the master block, and then add samples to the master block and mix. Discard the washing buffer and add 100 microliters of each diluted sample to the experimental blocks. For the negative control block, add 100 microliters of only the antibody diluent.Then, incubate the slides for one hour at room temperature with gentle shaking. Next, use 120 microliters of PBST to wash each block five times for three minutes each with shaking. Then add 100 microliters of biotinylated goat anti-Human antibody diluted in antibody diluent to each block.Cover the slides and incubate for one hour at room temperature with gentle shaking. Use 120 microliters of PBST to wash each block five times for three minutes each with shaking. Next, add 100 microliters of streptavidin SY5 conjugate diluted in 5%BSAT, in a ratio of one to two thousand to each block.Cover the slides with foil and incubate for 15 minutes at room temperature with gentle shaking. Wash the slides first with 120 microliters of PBST five times for one minute each, and then wash them two more times for one minute each with PBS. To dry the slides, spin them for five minutes at 300 times gravity.Turn the laser scanner on 30 minutes prior to scanning, to allow it to warm up. Then place the slide in the scanner with the printed surface facing up until the plate light turns solid green. Next, press the Settings button.On the first tab, insure that the Automatic scrolling and Barcode are unchecked. Then, set the Pixel size to 10, Scan Mode to Medium, Acquisition to 635 only, Acquisition Mode to Manual, gain to 20, and power to low. On the second tab, insure that the slide type is unlabeled.On the third tab, set the Focus setting to Auto focus. Press the import grid button to select the appropriate array list associated with the scanned image. Then, press the autofind button to align the grid to the spots on the slide.Finally, press the Quantification Process button to measure the fluorescence intensity of each spot. Then, save the results in an appropriate format for further analysis. In this protocol, a protein microarray is compared with ELISA to assess the reproducibility between these two techniques.The Spearman correlation coefficient analysis shows significant agreement between the microarray and ELISA for detecting IgG Anti-toxin A, and Anti-toxin B levels in serum samples. Intro assay and inter assay reproducibility of the protein micro array are calculated using the serum samples of seven patients. The samples analyzed on two different array slides against Toxin A, Toxin B, SLP001, SLP002, SLP027, Toxin B from Toxin B only producing strain CCUG, and pCDTb antigens reveal good reproducibility.A protein microarray is performed to detect an isotype-specific antibody response against Clostridium difficile toxins. The serum samples of patients and healthy individuals are used to detect the levels of IgG and IgA against Toxin A, Toxin B, Toxin B from a Clostridium difficile Toxin B only expressing strain, and precursor form of a B fragment of binary toxin. Once mastered, this technique can be done in seven hours.The quality control experiment, different parameters such as selection of appropriate surface chemistry, printing buffers, blocking buffers, and dilutions of serum samples and secondary antibodies should be evaluated before running samples. Printing a high-quality array requires a good understanding of the array or software to adjust the printing of parameters according to the experimental protocol. It is crucial to maintain optimum performance of the arrayer, and a dust-free environment throughout the experiment.This technique will pave the way for researchers in the field of immunology and molecular biology, in terms of characterizing humoral immune responses to infection, and discovering serodiagnostic antigens. Don't forget that working with infected blood samples and bacterial toxins can be hazardous. In general, in laboratory, safety and standard precautions should always be taken into consideration while performing this procedure.

Research

JoVE Journal

Immunology and Infection

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Antígeno Específico de la En Vivo Matar ensayo utilizando CFSE células diana marcadas

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Investigación

Inmunología e Infección

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

El uso de un ensayo de microarrays Pan-viral (Virochip) a la pantalla de muestras clínicas de los patógenos virales

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Celular del cáncer colorrectal superficie de la proteína perfiles con un microarray de anticuerpos y la multiplexación de fluorescencia

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Trans-vivo hipersensibilidad de tipo retardada Ensayo para Antigen Reglamento Específico

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

El cultivo y mantenimiento<em&gt; Clostridium difficile</em&gt; En un ambiente anaeróbico

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Imaging Whole-animal y de citometría de flujo Las técnicas para T CD8 + respuestas celulares Análisis de antígeno-específicos después de nanopartículas Vacunación

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Cefoperazona tratados modelo de ratón clínicamente relevante<em&gt; Clostridium difficile</em&gt; R20291 Strain

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

Detección y enriquecimiento de Rare células B específicas de antígeno para el análisis del fenotipo y la función

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Protocolo para Caracterizar los Cambios Morfológicos de<em&gt; Clostridium difficile</em&gt; En respuesta al tratamiento antibiótico

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

En Vivo Ensayo para la detección de la función citolítica de células T específicas de antígeno utilizando un modelo de vacunación

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...