Standardized Assessments of Neutralizing Antibodies for HIV/AIDS Vaccine Development

David C. Montefiori, Duke University Medical Center

This site provides detailed information for making standardized assessments of neutralizing antibodies against HIV, SIV and SHIV using molecularly cloned Env-pseudotyped viruses in an optimized and validated luciferase (Luc) reporter gene assay. Access is provided to assay protocols and macros currently in use at the Duke Central Reference Laboratory. This site also contains educational slides, tutorials and links to relevant publications and assay reagents. Special attention is given to HIV-1 reference strains that are available as molecularly cloned Env-pseudotyped viruses.

Powerpoint Presentations:

• Neutralizing Antibody Assays for HIV-1, SIV and SHIV: Recent Advances in Technology
  
  
• Design and Use of HIV-1 Reference Strains for Standardized Assessments of Vaccine-Elicited Neutralizing Antibody Responses

• Protocol for Measuring Neutralizing Antibodies Against HIV-1, SIV and SHIV Using a Luciferase Reporter Gene Assay in TZM-BL Cells
  
  
• User Guide To the Luminescence Macro
  
  
• User Guide to the TCID50 Macro
  
  
• User Guide to the TCID50 - Cutoff Macro
  
  
• Preparation and Titration of HIV-1 Env-Pseudotyped Viruses
  
  
• Heat-Inactivation of Plasma and Serum Samples
  
  
• Preparation of Cells for Detection of Mycoplasma Species
  
  
• HIV-1 Isolation by PBMC Co-Culture
  
  
• Preparation of Cell-Free Stocks of TCLA HIV-1 in Cell Lines
  
  
• Preparation of Cell-Free Stocks of HIV-1 in PBMC
  
  
• Neutralizing Antibody Assay in PBMC
  
  
• Determination of Optimal Concentration of DEAE-Dextran
  
  
• Assay Excel Macros (note: change the filetype to 'xlt' before using):
  
  
  • Screening for Neutralization at a Single Sample Dilution
    
    
  • Measurement of Neutralization Titers
    
    
  • Calculation of TCID50
    
    
  • Calculation of TCID50 - Cutoff
    
    

• Recommendations for the Design and Use of Standard Virus Panels To Assess Neutralizing Antibody Responses Elicited by Candidate Human Immunodeficiency Virus Type 1 Vaccines
  
  
• Human Immunodeficiency Virus Type 1 env Clones from Acute and Early Subtype B Infections for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies
  
  
• Genetic and Neutralization Properties of Subtype C Human Immunodeficiency Virus Type 1 Molecular env Clones from Acute and early Heterosexually Acquired Infections in Southern Africa
  
  

• Standard Reference Panel of Subtype B HIV-1 Env Clones (NIH AIDS Research and Reference Reagent Program)
  
  
• Subtype C HIV-1 Reference Panel of Env Clones (NIH AIDS Research and Reference Reagent Program)
  
  
• Registry of HIV-1 Env Pseudoviruses
  
  

• Clade B and C sequences gp120 alignment (fasta)
• Clade B and C sequence gp120 alignment (pdf)