Detection Assays for HIV Proteins

Mario A. Ostrowski1, Tae‐Wook Chun2, Bruce Cheseboro2, Sharilyn K. Stanley2, Michel Tremblay3

1 University of Toronto, Toronto, null, 2 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, 3 University of Laval, Quebec, null
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 12.5
DOI:  10.1002/0471142735.im1205s70
Online Posting Date:  January, 2006
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Abstract

This updated unit describes four assays for detection of HIV proteins including: (1) a quantitative ELISA, (2) a quantitative immunoblotting assay, (3) a qualitative immunofluorescence assay, and (4) a functional assay to measure virusā€associated reverse transcriptase activity as an indicator of viral production.

     
 
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Table of Contents

  • Basic Protocol 1: Antibody‐Sandwich ELISA for HIV Antigens
  • Alternate Protocol 1: Kinetic ELISA for HIV p24 Antigen
  • Alternate Protocol 2: “In‐House” Antibody‐Sandwich ELISA for HIV Antigens
  • Basic Protocol 2: Immunoblotting Assay for Detection of HIV Proteins
  • Basic Protocol 3: Immunofluorescence Assay (IFA) for HIV Proteins
  • Alternate Protocol 3: Intracellular Flow Cytometry for HIV Proteins
  • Support Protocol 1: Generation of Human Anti‐HIV Serum Stock
  • Basic Protocol 4: Assay for HIV Reverse Transcriptase Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Antibody‐Sandwich ELISA for HIV Antigens

  Materials
  • p24 ELISA kit (e.g., Coulter HIV p24 Ag Assay, cat. no. 626391) containing:
    • Concentrated wash buffer
    • HIV p24 antigen solution
    • Anti‐p24 antibody–coated 96‐well microtiter plate (with adhesive cover)
    • Lysing buffer
    • Biotinylated antibody solution
    • Streptavidin‐horseradish peroxidase conjugate solution (SA‐HRPO)
    • Tetramethylbenzidine (TMB) solution
    • Stop reagent: e.g., 2 M H 2SO 4 or other reagent that destroys peroxidase activity
  • Culture supernatants or sera to be tested, with negative controls
  • Individual and multichannel pipettors (10‐ to 200‐µl volume) and disposable tips
  • ELISA microtiter plate washer
  • Microtiter plate reader (450 nm)

Alternate Protocol 1: Kinetic ELISA for HIV p24 Antigen

  • p24 antigen kinetic standard (Coulter, cat. no. 6604594)
  • SA buffer (Coulter)

Alternate Protocol 2: “In‐House” Antibody‐Sandwich ELISA for HIV Antigens

  Materials
  • HIV‐1 p24 hybridoma 183‐H12‐5C (NIH AIDS Research and Reference Reagent Program, cat. no. 1513)
  • Complete IMDM medium with 10% FBS ( appendix 2A)
  • Feeder cells (optional): C57BL/10 mouse spleen cells (see unit 3.1)
  • Freezing medium (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A), 4°C and room temperature
  • Elution buffer: 0.2 M glycine⋅HCl, pH 2.5
  • Neutralizing buffer: 1.0 M Na 2HPO 4, dibasic
  • HIV p24 hybridoma 31‐90‐25 (ATCC; http://www.atcc.org/) or human HIV immune globulin (NIH AIDS Research Reference Reagent Program)
  • Biotinylation kit (EZ‐Link Sulfo‐NHS‐LC‐Biotin; Pierce, cat. no. 21335; also see unit 18.2)
  • 50 mg/ml bovine serum albumin (BSA, fraction V) in 0.1 M sodium azide
  • Coating buffer (see recipe)
  • PBS‐T: PBS ( appendix 2A) containing 0.05% (v/v) Tween 20
  • PBS‐T (see above) containing 1% (w/v) bovine serum albumin (BSA, fraction V)
  • p24 standards (Coulter, cat. no. 6604594)
  • Samples to be tested
  • Disruption buffer (see recipe)
  • Poly HRP40‐Steptavidin (Research Diagnostics Inc., cat. no. RDI‐PHRP40‐SA2)
  • TMB‐S substrate (Research Diagnostics Inc., cat. no. RDI‐TMBS‐1L)
  • 1 M H 3PO 4 ( appendix 2A)
  • 25‐cm2 and 175‐cm2 tissue culture flasks
  • 250‐ml conical centrifuge bottles
  • 3.0‐ml (10 × 38–mm) affinity purification column containing recombinant Protein G–Sepharose 4B (Zymed, cat. no. 10‐1242); store in cold room at 4°C
  • UV‐Vis spectrophotometer
  • Dialysis tubing, MWCO 10,000 to 20,000 ( appendix 3H)
  • Concentration apparatus: Centricon 10 or Amicon system (e.g., Amicon Ultra‐15 for 300,000 kDa; Millipore, cat no. UFC903008)
  • 96‐well ELISA plates: IMMULON 2 plates (Dynatech Labs) or Immuno MaxiSorp plates (Nunc)
  • ELISA microtiter plate washer
  • Microtiter plate reader (450 nm filter; reference wavelength 690 nm)
  • Additional reagents and equipment for production of hybridoma supernatants (unit 2.6), purification of monoclonal antibodies (unit 2.7), dialysis and concentration of proteins ( appendix 3H), and biotinylation of monoclonal antibodies (unit 18.2)

Basic Protocol 2: Immunoblotting Assay for Detection of HIV Proteins

  Materials
  • Cells to be tested
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • TENC lysing buffer (see recipe)
  • TENC lysing buffer/2% sodium deoxycholate (NaDOC)
  • Transfer buffer (see recipe)
  • 5% nonfat dry milk/1× TN buffer, pH 7.4 (see recipe)
  • TN‐TN buffer, pH 7.4
  • Anti‐HIV polyclonal serum (e.g., protocol 7)
  • 3% (v/v) bovine serum albumin (BSA)/TN‐T buffer
  • TN‐T buffer, pH 7.4 (see recipe)
  • 0.1 mCi/ml [125I]protein A (low‐specific‐activity; DuPont NEN, cat. no. NEX146‐L) in 3% BSA/TN‐T buffer
  • 10 mM EDTA ( appendix 2A)/TN‐TN buffer
  • 1× TN buffer, pH 7.4 (see recipe)
  • Tabletop centrifuge
  • Glass dish or sealable bag
  • Platform rocker
  • Additional reagents and equipment for counting cells ( appendix 3B), one‐dimensional denaturing gel electrophoresis (unit 8.4), electrophoretic transfer of proteins to nitrocellulose (immunoblotting; unit 8.10), preparation of serum from blood (unit 2.4), and autoradiography ( appendix 3J)

Basic Protocol 3: Immunofluorescence Assay (IFA) for HIV Proteins

  Materials
  • Fixative solution: 1:1 (v/v) methanol/acetone
  • Cells to be tested
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • Bleach
  • 85% to 95% ethanol
  • Human polyclonal anti‐HIV serum ( protocol 7)
  • Human serum from an uninfected source
  • Rhodamine (Sigma)
  • FITC‐conjugated goat anti‐human IgG or other FITC‐conjugated anti‐human IgG (unit 5.3 or commercial supplier)
  • Glycerol
  • Permount (e.g., Fisher)
  • Glass fixative dish
  • Tabletop centrifuge and appropriate tubes
  • Glass slides (preferably frosted on one end)
  • Metal slide holder for fixing
  • Cytospin‐3 centrifuge with chambers, cardboard mounts, and metal holder for slides (Shandon/Lipshaw)
  • Wax pencil
  • Platform rocker
  • Fluorescence microscope with green and red filters for FITC and rhodamine
  • Staining chamber (see step annotation)
  • Additional reagents and equipment for cell counting ( appendix 3A)

Alternate Protocol 3: Intracellular Flow Cytometry for HIV Proteins

  Materials
  • Cells to be tested
  • FACS wash buffer, cold: PBS ( appendix 2A)/1% fetal bovine serum/0.09% (w/v) NaN 3
  • Cytofix/Cytoperm Plus kit (BD Biosciences, cat no. 554714) or equivalent
  • Anti‐HIV Fluorochrome conjugated antibody, KC57 (Coulter no. 6604665 is FITC‐conjugated; 6604667 is RD1 conjugated, detected in FL2 window)
  • Isotype control: IgG 1κ (mouse, clone MOPC‐21), fluorochrome conjugated, (BD Biosciences, cat no. 554679 for FITC; cat no. 55136 for PE)
  • 1% paraformaldehyde/PBS solution

Support Protocol 1: Generation of Human Anti‐HIV Serum Stock

  Materials
  • Cell cultures to be assayed
  • Cell culture supernatant known to contain HIV (positive control)
    • RT master mix (500 ml; see Table 12.5.2)
  • 10 mCi/ml [α−32P]dTTP (>400 Ci/mmol; DuPont NEN, cat. no. BLU‐505H)
  • 1 M dithiothreitol (DTT; store in 100‐µl aliquots at −20°C.
  • 1× SSC (unit 10.6)
  • 85% to 95% ethanol
  • Biosafe II Scintillation Cocktail (Research Products International, cat. no., 111195)
  • 96‐well U‐ or V‐bottom microtiter plate (can be nonsterile)
  • Multichannel pipettor
  • DEAE Filtermat paper (Wallac‐PE Life Sciences, cat. no. 1450‐522)
  • Dish
  • Platform rocker
  • Plastic bags (sheaths) for filter papers (Wallac‐PE Life Sciences, cat. no. 1450‐432)
  • Beta counter (e.g., 1450 Wallac Microbeta Trilux Scintillation counter, model no. 1450‐012)
  • Additional reagents and equipment for autoradiography ( appendix 3J)
CAUTION: Radioactive materials require special handling. See appendix 1Q and the institutional Radiation Safety Office for guidelines concerning proper handling and disposal.
Table 2.5.2   Materials   Reverse Transcriptase (RT) Master Mix b   Reverse Transcriptase (RT) Master Mix

Reagent Volume Concentration in cocktail Final concentration in reaction mix
Distilled H 2O 419.5 ml
1 M Tris⋅Cl, pH 7.8 c 30.0 ml 60 mM 50 mM
1 M KCl 37.5 ml 75 mM 63 mM
1 M MgCl 2 2.5 ml 5 mM 4.2 mM
10% Nonidet P‐40 5.0 ml 0.1% 0.08%
0.5 M EDTA 1.020 ml 2.04 mM 1.7 mM
2 mg/ml Poly(A) d 1.250 ml 5 µg/ml 4.2 µg/ml
25 µg/ml Oligo‐dT e 3.25 ml 0.16 µg/ml 0.14 µg/ml

 bRT master mix can be made up ahead of time and stored in 50‐ml aliquots at −20° or −70°C indefinitely. Frozen aliquots can be thawed in a 37°C water bath prior to use.
 cSee recipe in appendix 2A.
 dAmersham Biosciences, cat. no. 27‐4110‐01.
 eAmersham Biosciences, cat. no. 27‐7858‐02.
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Literature Cited

Literature Cited
   Burnette, W.H. 1981. Western blotting: Electrophoretic transfer of proteins from SDS‐polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112:195‐203.
   Bounou, S., Leclerc, J.E., and Tremblay, M.J. 2002. The presence of host ICAM‐1 in laboratory and clinical strains of HIV‐1 increases virus infectivity and CD4+ T‐cell depletion in human lymphoid tissue, a major site of replication in vivo. J. Virol. 76:1004‐1014.
   Centers for Disease Control. 1989. Interpretation and use of the Western blot assay for serodiagnosis of human immunodeficiency virus type 1 infections. Morb. Mortal. Wkly. Rep. 38:1‐7.
   Clouse, K.A., Powell, D., Washington, I., Poli, G., Strebel, K., Farrar, W., Barstad, P., Kovacs, J., Fauci, A.S., and Folks, T.M. 1989. Monokine regulation of human immunodeficiency virus‐1 expression in a chronically infected human T cell clone. J. Immunol. 142:431‐438.
   Davey, R.T. Jr. and Lane, H.C. 1990. Laboratory methods in the diagnosis and prognostic staging of infection with human immunodeficiency virus type 1. Rev. Infect. Dis. 12:912‐930.
   Fernie, B.F., Poli, G., and Fauci, A.S. 1991. Alpha interferon suppresses virion but not soluble human immunodeficiency virus antigen production in chronically infected T‐lymphocytic cells. J. Virol. 65:3968‐3971.
   Gallo, R.C., Sarin, P.S., Gelmann, E.P., Robert‐Guroff, M., Richardson, E., Kalyanaraman, V.S., Mann, D., Sidhu, G.D., Stahl, R.E., Zolla‐Pazner, S., Leibowitch, J., and Popovic, M. 1983. Isolation of human T‐cell leukemia virus in acquired immune deficiency syndrome (AIDS). Science 220:865‐867.
   Nishanian, P., Huskins, K.R., Stehn, S., Detels, R., and Fahey, J.L. 1990. A simple method for improved assay demonstrates that HIV p24 antigen is present as immune complexes in most sera from HIV‐infected individuals. J. Infect. Dis. 162:21‐28.
   Watkins, S. 1989. Immunohistochemistry. In Current Protocols in Molecular Biology (F.M. Aubusel, R. Brent, R.E. Kingston, D.D. Moore, J. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 14.6.1‐14.6.13. John Wiley & Sons, Hoboken, N.J.
   Wehrly, K. and Chesebro, B. 1997. p24 antigen capture assay for quantification of Human Immunodeficiency Virus using readily available inexpensive reagents. Methods Enzymol. 12:288‐293.
   Willey, R.L., Smith, D.H., Lasky, L.A., Theodore, T.S., Earl, P.L., Moss, B., Capen, D., and Martin, M.A. 1988. In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity. J. Virol. 62:139‐146.
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