Evaluating Neutralizing Antibodies Against HIV, SIV, and SHIV in Luciferase Reporter Gene Assays

David C. Montefiori1

1 Duke University Medical Center, Durham, North Carolina
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 12.11
DOI:  10.1002/0471142735.im1211s64
Online Posting Date:  January, 2005
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Abstract

Neutralizing antibody assays are powerful tools for assessing humoral immunity in AIDS virus infection and vaccine development. Although many different assays have been described, all are based on the same principle, measuring reductions in virus infectivity. This unit describes two assays utilizing a genetically engineered cell lines that are susceptible to infection by most strains of HIV‐1, SIV, and SHIV. One assay is designed for optimal performance with uncloned viruses produced in either PBMC or CD4+ T cell lines. A second assay is designed for single‐cycle infection with molecularly cloned pseudoviruses produced by transfection in 293T cells. Both assays are performed in a 96‐well format and use tat‐responsive luciferase reporter gene expression as readout.

Keywords: Neutralization; Antibodies; HIV; AIDS; Luciferase

     
 
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Table of Contents

  • Basic Protocol 1: Multiple‐Round Infection Assay in 5.25EGFP.Luc.M7 Cells for Uncloned Viruses
  • Basic Protocol 2: Single‐Round Infection Assay in TZM‐bl Cells for Molecularly Cloned Pseudoviruses
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Multiple‐Round Infection Assay in 5.25EGFP.Luc.M7 Cells for Uncloned Viruses

  Materials
  • Cell‐free virus stocks of HIV‐1, SIV, and SHIV: seed stocks available from the NIH AIDS Research and Reference Reagent Program ( ); stocks should be expanded in PHA‐ stimulated PBMC from HIV‐seronegative (normal) donors; alternatively, T  cell–line adapted (TCLA) strains of virus may be expanded in human CD4+ lymphoblastoid cell lines (see unit 12.2 for both techniques)
  • 5.25.EGFP.Luc.M7 cells: may be obtained from the Salk Institute by contacting either Pamela Bock ( ) or Dr. Nathaniel R. Landau ( )
  • DEAE‐CM1 (prepared with complete RPMI‐1640 medium; see recipe)
  • Bright‐Glo luciferase assay system (Promega)
  • Serum or plasma samples to be tested for neutralization antibodies
  • Positive control: serum or plasma with known neutralization titer against target virus
  • Negative controls: e.g., corresponding preimmune or preinfection samples from each study subject
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • 96‐well sterile flat‐bottom culture plates, low‐evaporation (Corning Costar no. 3595, available from VWR)
  • 50‐ml sterile reagent reservoirs (Falcon)
  • 96‐well white solid flat‐bottom plates (Corning Costar no. 3912; available from Fisher)
  • Multiscan MCC/340 luminometer, Model 347 (MTX Lab Systems) or equivalent
  • 56°C water bath
  • Sterile gauze pads
  • Fluorescence microscope equipped to detect GFP (Olympus CKX41 or equivalent)
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A)

Basic Protocol 2: Single‐Round Infection Assay in TZM‐bl Cells for Molecularly Cloned Pseudoviruses

  Materials
  • Env‐pseudotyped virus (see recipe)
  • DEAE‐CM2 (prepared with complete DMEM medium; see recipe)
  • TZM‐bl cells (NIH AIDS Research and Reference Reagent Program; ) growing in 75‐cm2 flasks
  • Bright‐Glo luciferase assay system (Promega)
  • Serum or plasma samples to be tested for neutralization antibodies
  • Complete DMEM medium containing 10% heat‐inactivated FBS ( appendix 2A)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.25% trypsin/1 mM EDTA, sterile (Life Technologies)
  • 96‐well sterile flat‐bottom culture plates, low‐evaporation (Corning Costar no. 3595, available from VWR)
  • 50‐ml sterile reagent reservoirs (Falcon)
  • 96‐well black solid flat‐bottom plates (Corning Costar no. 3915, available from VWR)
  • Multiscan MCC/340 luminometer, Model 347 (MTX Lab Systems) or equivalent
  • 56°C water bath
  • Sterile gauze pads
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
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