Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1

The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used f...

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Published in	Journal of immunological methods Vol. 409; pp. 131 - 146
Main Authors	Sarzotti-Kelsoe, Marcella, Bailer, Robert T., Turk, Ellen, Lin, Chen-li, Bilska, Miroslawa, Greene, Kelli M., Gao, Hongmei, Todd, Christopher A., Ozaki, Daniel A., Seaman, Michael S., Mascola, John R., Montefiori, David C.
Format	Journal Article
Language	English
Published	Netherlands Elsevier B.V 01.07.2014
Subjects	Antibodies, Neutralizing - blood Assay validation Automation, Laboratory - standards Biomarkers - blood clinical trials compliance detection limit GCLP gene expression Guideline Adherence - standards guidelines HeLa Cells High-Throughput Screening Assays - standards HIV HIV Antibodies - blood HIV Infections - blood HIV Infections - diagnosis HIV Infections - immunology HIV-1 - genetics HIV-1 - immunology Human immunodeficiency virus 1 Humans Limit of Detection luciferase neutralization neutralization tests Neutralization Tests - standards neutralizing antibodies Neutralizing antibody Observer Variation Practice Guidelines as Topic - standards Predictive Value of Tests Quality Control reporter genes Reproducibility of Results Time Factors Transfection TZM-bl cells viruses TZM-bl cells GCLP HIV Assay validation Neutralizing antibody
Online Access	Get full text
ISSN	0022-1759 1872-7905 1872-7905
DOI	10.1016/j.jim.2013.11.022

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Abstract	The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of pre-clinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format.
AbstractList	The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of pre-clinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format. The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of preclinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format. The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of pre-clinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format.The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of pre-clinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format.
Author	Sarzotti-Kelsoe, Marcella Seaman, Michael S. Ozaki, Daniel A. Bailer, Robert T. Lin, Chen-li Bilska, Miroslawa Greene, Kelli M. Montefiori, David C. Mascola, John R. Todd, Christopher A. Turk, Ellen Gao, Hongmei
AuthorAffiliation	3 Vaccine Research Center, National Institutes of Health (NIH), Bethesda, MD 1 Department of Immunology, Duke University Medical Center, Durham, NC 4 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 2 Department of Surgery, Duke University Medical Center, Durham, NC
AuthorAffiliation_xml	– name: 1 Department of Immunology, Duke University Medical Center, Durham, NC – name: 2 Department of Surgery, Duke University Medical Center, Durham, NC – name: 4 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA – name: 3 Vaccine Research Center, National Institutes of Health (NIH), Bethesda, MD
Author_xml	– sequence: 1 givenname: Marcella surname: Sarzotti-Kelsoe fullname: Sarzotti-Kelsoe, Marcella email: marcella.sarzottikelsoe@dm.duke.edu organization: Department of Immunology, Duke University Medical Center, Durham, NC, United States – sequence: 2 givenname: Robert T. surname: Bailer fullname: Bailer, Robert T. organization: Vaccine Research Center, National Institutes of Health (NIH), Bethesda, MD, United States – sequence: 3 givenname: Ellen surname: Turk fullname: Turk, Ellen organization: Vaccine Research Center, National Institutes of Health (NIH), Bethesda, MD, United States – sequence: 4 givenname: Chen-li surname: Lin fullname: Lin, Chen-li organization: Vaccine Research Center, National Institutes of Health (NIH), Bethesda, MD, United States – sequence: 5 givenname: Miroslawa surname: Bilska fullname: Bilska, Miroslawa organization: Department of Surgery, Duke University Medical Center, Durham, NC, United States – sequence: 6 givenname: Kelli M. surname: Greene fullname: Greene, Kelli M. organization: Department of Surgery, Duke University Medical Center, Durham, NC, United States – sequence: 7 givenname: Hongmei surname: Gao fullname: Gao, Hongmei organization: Department of Surgery, Duke University Medical Center, Durham, NC, United States – sequence: 8 givenname: Christopher A. surname: Todd fullname: Todd, Christopher A. organization: Department of Surgery, Duke University Medical Center, Durham, NC, United States – sequence: 9 givenname: Daniel A. surname: Ozaki fullname: Ozaki, Daniel A. organization: Department of Surgery, Duke University Medical Center, Durham, NC, United States – sequence: 10 givenname: Michael S. surname: Seaman fullname: Seaman, Michael S. organization: Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States – sequence: 11 givenname: John R. surname: Mascola fullname: Mascola, John R. organization: Vaccine Research Center, National Institutes of Health (NIH), Bethesda, MD, United States – sequence: 12 givenname: David C. surname: Montefiori fullname: Montefiori, David C. organization: Department of Surgery, Duke University Medical Center, Durham, NC, United States
BackLink	https://www.ncbi.nlm.nih.gov/pubmed/24291345$$D View this record in MEDLINE/PubMed
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Snippet	The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene...
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SubjectTerms	Antibodies, Neutralizing - blood Assay validation Automation, Laboratory - standards Biomarkers - blood clinical trials compliance detection limit GCLP gene expression Guideline Adherence - standards guidelines HeLa Cells High-Throughput Screening Assays - standards HIV HIV Antibodies - blood HIV Infections - blood HIV Infections - diagnosis HIV Infections - immunology HIV-1 - genetics HIV-1 - immunology Human immunodeficiency virus 1 Humans Limit of Detection luciferase neutralization neutralization tests Neutralization Tests - standards neutralizing antibodies Neutralizing antibody Observer Variation Practice Guidelines as Topic - standards Predictive Value of Tests Quality Control reporter genes Reproducibility of Results Time Factors Transfection TZM-bl cells viruses
Title	Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1
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